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Repetitive Elements in the 5′ Untranslated Region of a Human Zinc-Finger Gene Modulate Transcription and Translation Efficiency
Abstract
A substantial proportion of the human genome consists of repetitive sequences. Although most of these sequences are nonessential for the organism, retroelements, such as Alu sequences, L1s, and HERVs (human endogenous retroviruses), have recently been implicated in the regulation of various genes. Our laboratory previously identified a novel, alternatively spliced zinc-finger gene, ZNF177, which incorporates Alu L1, and HERV segments into the 5' untranslated region (UTR) of transcripts. In this study, we investigated the genomic structure and functional significance of the repetitive sequences in the 5' UTR of ZNF177 mRNAs. Using luciferase and GFP reporter constructs, we assessed the effect of the HERV, Alu, and L1 sequences on gene expression levels. Our results indicate that the presence of the retroelement sequences, particularly the Alu and L1 segments which form one 5' UTR exon, modifies the expression level of both reporter genes. We present evidence that the Alu and L1 sequences alter both the RNA and protein levels of reporter genes by increasing transcription efficiency while decreasing translation efficiency. Our findings indicate that the Alu and L1 repeats in the 5' UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation of the zinc finger gene. In addition, our analysis of a 5' UTR database suggests that 4% of human 5' UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats. These results illustrate the complex regulatory effects that retroelements can have on human gene expression.
... Human-specific HERV-K LTRs generate antisense transcripts to SLC4A8 and IFT172 mRNAs [128] REs as insulator elements B2 SINE element located in the murine growth hormone locus serves as a boundary to block the influence of repressive chromatin modifications [134]; Drosophila LTR retrotransposon gypsy in the 5 0 region of the gene yellow blocks the action of the upstream located enhancers and is responsible for the pigmentation of cuticula [137] REs as regulators of translation Alu and L1 segments in the 5 0 UTR of human ZNF177 gene modify gene expression on the protein level by decreasing translation efficiency [140] There are numerous reported cases of human diseases caused by recombination between REs. For example, glycogen storage disease [44], Alport syndrome [45] as a result of recombination between L1 elements, and complete germ cell aplasia due to recombination between HERV-I [46]. ...
... The presence of the Alu and L1 segments which form one 5 0 UTR exon modifies gene expression on the protein level by decreasing translation efficiency. Interestingly, the same Alu and L1 repeats in the 5 0 UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation [140]. Approximately 4% of human 5 0 UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats [140]. ...
... Interestingly, the same Alu and L1 repeats in the 5 0 UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation [140]. Approximately 4% of human 5 0 UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats [140]. In the mouse genome, there is a SINE retrotransposon-derived gene for neuronal dendrite-specific BC1 RNA. ...
Retroelements comprise a considerable fraction of eukaryotic genomes. Since their initial discovery by Barbara McClintock in maize DNA, retroelements have been found in genomes of almost all organisms. First considered as a "junk DNA" or genomic parasites, they were shown to influence genome functioning and to promote genetic innovations. For this reason, they were suggested as an important creative force in the genome evolution and adaptation of an organism to altered environmental conditions. In this review, we summarize the up-to-date knowledge of different ways of retroelement involvement in structural and functional evolution of genes and genomes, as well as the mechanisms generated by cells to control their retrotransposition.
... The presence of the Alu and L1 segments which form one 5 0 -UTR exon modifies gene expression on the protein level by decreasing translation efficiency. Interestingly, the same Alu and L1 repeats in the 5 0 -UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation (Landry et al., 2001). Approximately 4% of human 5 0 -UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats (Landry et al., 2001). ...
... Interestingly, the same Alu and L1 repeats in the 5 0 -UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation (Landry et al., 2001). Approximately 4% of human 5 0 -UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats (Landry et al., 2001). In the mouse genome, there is a SINE retrotransposon-derived gene for neuronal dendrite-specific BC1 RNA. ...
Repetitive sequences occupy a huge fraction of essentially every eukaryotic genome. Repetitive sequences cover more than 50% of mammalian genomic DNAs, whereas gene exons and protein-coding sequences occupy only ~3% and 1%, respectively. Numerous genomic repeats include genes themselves. They generally encode "selfish" proteins necessary for the proliferation of transposable elements (TEs) in the host genome. The major part of evolutionary "older" TEs accumulated mutations over time and fails to encode functional proteins. However, repeats have important functions also on the RNA level. Repetitive transcripts may serve as multifunctional RNAs by participating in the antisense regulation of gene activity and by competing with the host-encoded transcripts for cellular factors. In addition, genomic repeats include regulatory sequences like promoters, enhancers, splice sites, polyadenylation signals, and insulators, which actively reshape cellular transcriptomes. TE expression is tightly controlled by the host cells, and some mechanisms of this regulation were recently decoded. Finally, capacity of TEs to proliferate in the host genome led to the development of multiple biotechnological applications.
... The movement of TEs are an important source of variation within a genome, wherein TEs can alter gene expression [79,80], disrupt coding genes [81], or promote recombination [82], allowing for dramatic and rapid restructuring of the genome that may exceed the changes offered by point mutations [83] ( Figure 2III). These changes may allow populations to explore a fitness landscape more fully in a shorter period of time, increasing the adaptability of the population [84][85][86]. ...
Chromosome reshuffling events are often a foundational mechanism by which speciation can occur, giving rise to highly derivative karyotypes even amongst closely related species. Yet, the features that distinguish lineages prone to such rapid chromosome evolution from those that maintain stable karyotypes across evolutionary time are still to be defined. In this review, we summarize lineages prone to rapid karyotypic evolution in the context of Simpson’s rates of evolution—tachytelic, horotelic, and bradytelic—and outline the mechanisms proposed to contribute to chromosome rearrangements, their fixation, and their potential impact on speciation events. Furthermore, we discuss relevant genomic features that underpin chromosome variation, including patterns of fusions/fissions, centromere positioning, and epigenetic marks such as DNA methylation. Finally, in the era of telomere-to-telomere genomics, we discuss the value of gapless genome resources to the future of research focused on the plasticity of highly rearranged karyotypes.
... Also, insertion of TEs into intronic regions can have deleterious effects, as insertion of Alu elements and LINE-1 within an intron can introduce novel splice sites, leading to alternative splicing events that disrupt transcriptional integrity [36,37]. Some studies have also shown that insertion of TEs into 5 or 3 regions of genes may disrupt normal gene expression [38]. Therefore, the collective impact of alterations in gene expression due to TE insertions has been associated with various disease conditions such as cancer and genetic disorders [39]. ...
Most living organisms have in their genome a sizable proportion of DNA sequences capable of mobilization; these sequences are commonly referred to as transposons, transposable elements (TEs), or jumping genes. Although long thought to have no biological significance, advances in DNA sequencing and analytical technologies have enabled precise characterization of TEs and confirmed their ubiquitous presence across all forms of life. These findings have ignited intense debates over their biological significance. The available evidence now supports the notion that TEs exert major influence over many biological aspects of organismal life. Transposable elements contribute significantly to the evolution of the genome by giving rise to genetic variations in both active and passive modes. Due to their intrinsic nature of mobility within the genome, TEs primarily cause gene disruption and large-scale genomic alterations including inversions, deletions, and duplications. Besides genomic instability, growing evidence also points to many physiologically important functions of TEs, such as gene regulation through cis-acting control elements and modulation of the transcriptome through epigenetic control. In this review, we discuss the latest evidence demonstrating the impact of TEs on genome stability and the underling mechanisms, including those developed to mitigate the deleterious impact of TEs on genomic stability and human health. We have also highlighted the potential therapeutic application of TEs.
... In contrast, our knowledge about the contribution of transposons to the genesis and evolution of human uORFs is still limited. Among human 5′UTRs, 4% harbor Alu elements, the major subfamily of human SINEs, which might have substantial influence on gene expression, as evidenced by a luciferase reporter assay for transposons in the 5′UTR of ZNF177 (Landry, Medstrand, & Mager, 2001). In addition, 287 human uORFs stem from Alu elements that were incorporated into the 5′UTRs of human genes, mainly by exonization (Shen et al., 2011). ...
Transposons are major drivers of mammalian genome evolution. To obtain new insights into the contribution of transposons to the regulation of protein translation, we here examined how transposons affected the genesis and function of upstream open reading frames (uORFs), which serve as cis-acting elements to regulate translation from annotated ORFs (anORFs) located downstream of the uORFs in eukaryotic mRNAs. Among 39,786 human uORFs, 3,992 had ATG trinucleotides of a transposon origin, termed “transposon-derived upstream ATGs” or TuATGs. Luciferase reporter assays suggested that many TuATGs modulate translation from anORFs. Comparisons with transposon consensus sequences revealed that most TuATGs were generated by nucleotide substitutions in non-ATG trinucleotides of integrated transposons. Among these non-ATG trinucleotides, GTG and ACG were converted into TuATGs more frequently, indicating a CpG methylation-mediated process of TuATG formation. Interestingly, it is likely that this process accelerated human-specific upstream ATG formation within transposon sequences in 5′ untranslated regions after divergence between human and nonhuman primates. Methylation-mediated TuATG formation seems to be ongoing in the modern human population and could alter the expression of disease-related proteins. This study shows that transposons have potentially been shaping the human proteome landscape via cis-acting uORF creation.
... Hypomethylation of Alu elements was generally associated with decreased protein synthesis of nearby genes (cf. Landry et al, 2001), presumably via their function as genomic elements or as transcribed RNA (Wang and Huang, 2014). The AluJb element in the SLC6A4 promoter studied here belongs to the evolutionary early subfamily of AluJb and contains, as expected for this group, numerous transcriptionfactor-binding sites, particularly that for the repressive PAX6 (Dannlowski et al, 2014). ...
DNA methylation profiles of the serotonin transporter gene (SLC6A4) have been shown to alter SLC6A4 expression, drive antidepressant treatment response and modify brain functions. This study investigated whether methylation of an AluJb element in the SLC6A4 promotor was associated with major depressive disorder (MDD), amygdala reactivity to emotional faces, 5-HTTLPR/rs25531 polymorphism, and recent stress. MDD patients (n=122) and healthy controls (HC, n=176) underwent fMRI during an emotional face-matching task. Individual SLC6A4 AluJb methylation profiles were ascertained and associated with MDD, amygdala reactivity, 5-HTTLPR/rs25531, and stress. SLC6A4 AluJb methylation was significantly lower in MDD compared to HC and in stressed compared to less stressed participants. Lower AluJb methylation was particularly found in 5-HTTLPR/rs25531 risk allele carriers under stress and correlated with less depressive episodes. fMRI analysis revealed a significant interaction of AluJb methylation and diagnosis in the amygdala, with MDD patients showing lower AluJb methylation associated with decreased amygdala reactivity. While no joint effect of AluJb methylation and 5-HTTLPR/rs25531 existed, risk allele carriers showed significantly increased bilateral amygdala activation. These findings suggest a role of SLC6A4 AluJb methylation in MDD, amygdala reactivity, and stress reaction, partly interwoven with 5-HTTLPR/rs25531 effects. Patients with low methylation in conjunction with a shorter MDD history and decreased amygdala reactivity might feature a more stress-adaptive epigenetic process, maybe via theoretically possible endogenous antidepressant-like effects. In contrast, patients with higher methylation might possibly suffer from impaired epigenetic adaption to chronic stress. Further, the 5-HTTLPR/rs25531 association with amygdala activation was confirmed in our large sample.
... Gene function can be affected by LINE-1 retrotransposition when the gene coding region is the target site of insertion. However, the ability of these retrotransposons to alter gene expression without interfering with the coding region was also documented (see Landry et al. 2001;Waterston et al. 2002). LINE-1 insertion in the UTR regions of a gene can affect the regulation of its transcription and translation. ...
One of the clearest evidences that emerged from the eukaryotic genome sequencing projects was the high content in repetitive DNA sequences that these genomes harbour, being the extraordinary genome size variation found between taxa attributed to the differential amplification and deletion of these sequence families. However, despite its abundance, the role(s) that these sequences play in the genomes has always been shrouded in great mystery, as unlike genes, it was never assigned to them the ability to code proteins (at least proteins that are not involved in their own replication and genomic integration, as is the case of Transposable Elements). For this reason, these sequences were initially designated as “junk” DNA, with no function assigned. Presently, these sequences have won the deserved respect and are now regarded as a crucial fraction of eukaryotic genomes, recognized as important regulatory elements and also as being implicated in the occurrence of chromosomal rearrangements, with an important role in genome evolution. This was, precisely, the main goal of this work: to contribute to the understanding of the repetitive sequences significance in the evolution of eukaryotic genomes. For this purpose, it was analysed the repetitive genomic fraction of five Cricetidae/Muridae Rodentia species, with very distinct karyotypes, regarding tandem and dispersed repeats: Satellite DNAs (satDNAs), Interstitial Telomeric Sequences (ITSs) and LINE-1 Retrotransposons. A detailed analysis about the distribution and molecular nature of the Constitutive Heterochromatin (CH) of these rodent genomes was also performed.
The integration of all data allowed to understand how the five studied genomes evolved and to reconstruct the chromosomal evolutionary events elapsed, where the repetitive sequences were unquestionably involved. Indeed, all the results obtained here converge to this same conclusion. Namely, a strong association was observed between both the distribution and the level of CH heterogeneity with the evolutionary pathway that these karyotypes followed. In fact, for two of these species, a detailed analysis on the location of evolutionary breakpoint and CH regions revealed a very high coincidence between them. Other works focused on the evolution of the other three species, reported a similar relationship. Therefore, the repeats located in heterochromatin seem to be highly involved in the occurrence of chromosomal rearrangements, either by promoting directly chromosome reorganizations and/or because correspond to fragile regions prone to chromosome breakage. The analysis of the repeats located in CH regions performed here, namely satDNAs (exclusively heterochromatic), ITSs (mainly located in CH regions) and LINE-1 (frequently located in CH), really suggest the repetitive sequences as a driving force in the occurrence of chromosomal rearrangements. The repetitive nature per se of the different classes of repeats studied favours recombinational events between homologous sequences in non-homologous regions, which may culminate in chromosomal restructurings. Nevertheless the role in the origin of chromosomal reorganizations is particularly proposed for satDNAs, due to its characteristic evolutionary mode (concerted evolution) generally marked by rapid sequence mutations, copy number variations and/or intragenomic movements, driven by different recombinational events (as unequal crossing-over or rolling circle replication/reinsertion), that may induce chromosome breakage.
Additionally, beyond its important function in genome restructuring, the data obtained in this work also suggest other roles to repetitive sequences. An analysis devoted to the transcriptional activity of some of the studied satDNAs supports the role of these sequences in many other functions, as in control of gene expression, chromatin remodelation, cellular response to stress and centromeric function. LINE-1 sequences as well have important functions in control of gene expression, acting in gene imprinting and in X-chromosome inactivation. Thereby, despite initially considered useless genomic elements, in the light of all this data, it is impossible to deny that repetitive sequences are crucial for proper functioning and evolution of eukaryotic genomes, dethroning to our view the importance given in the past just to the protein-coding sequences. It is really now difficult to understand how these sequences, so abundant in eukaryotic genomes, may have been considered unnecessary, just because a coding capacity was not reported. After all, the protein-coding sequences only account for a tiny part of genomes (~1,5% of the human genome).
The present thesis resulted in the elaboration of seven articles that are published, submitted or in preparation for submission to indexed international scientific journals.
KEY WORDS: Repetitive DNA Sequences, Chromosomal Evolution, Tandem Repeats, Long Interspersed Nuclear Elements-1, Rodentia
... 37 HERV proviruses can also downregulate translation of a nearby host gene, presumably through strong secondary structure. 38,39 Thus, a price to be paid for the genetic flexibility provided by ERVs is a small pathogenic potential remaining even after many millions of years. ...
E ndogenous retroviruses (ERVs) are remnants of retroviral infections. ERVs preserve functions of exogenous retroviruses to various extents. ERVs are both parasites and symbionts. Although the most pathogenic elements are eliminated by selection, some pathogenicity may remain. Some recently endogenized elements of mice and cats are known to cause disease. The situation in humans is less certain. Diseases where a role for ERVs has been discussed are multiple sclerosis, schizophrenia, diabetes, systemic lupus erythematosus, semi-noma, malignant melanoma, preeclampsia and azoospermia. Several pathogenic mechanisms have been implicated: Antigenic mimicry, immune dysregulation, receptor interference, growth stimulation by cis-or transactivation, loss of physiological functions mediated by retroviral genes, and gene loss by illegitimate recombination are among them. In most cases, much work remains before a pathogenic mechanism is established. The biology of HERVs must be better understood in order to understand their role in human disease.
... Furthermore, L1 promoters contain binding sites for various transcription factors and regulatory proteins that can alter the gene expression in response to various stimuli[242- 245]. L1 sequences can exert their influence on the host gene expression by altering the promoter specificity or strength[246][247][248][249][250]. ...
We describe an as yet unreported neocentric small supernumerary marker chromosome (sSMC) derived from chromosome 1p21.3p21.2. It was present in 80% of the lymphocytes in a male patient with intellectual disability, severe speech deficit, mild dysmorphic features, and hyperactivity with elements of autism spectrum disorder (ASD).
Several important neurodevelopmental genes are affected by the 3.56 Mb copy number gain of 1p21.3p21.2, which may be considered reciprocal in gene content to the recently recognized 1p21.3 microdeletion syndrome. Both 1p21.3 deletions and the presented duplication display overlapping symptoms, fitting the same disorder category. Contribution of coding and non-coding genes to the phenotype is discussed in the light of cellular and intercellular homeostasis disequilibrium. In line with this the presented 1p21.3p21.2 copy number gain correlated to 1p21.3 microdeletion syndrome verifies the hypothesis of a cumulative effect of the number of deregulated genes - homeostasis disequilibrium leading to overlapping phenotypes between microdeletion and microduplication syndromes.
Although miR-137 appears to be the major player in the 1p21.3p21.2 region, deregulation of the DPYD (dihydropyrimidine dehydrogenase) gene may potentially affect neighboring genes underlying the overlapping symptoms present in both the copy number loss and copy number gain of 1p21. Namely, the all-in approach revealed that DPYD is a complex gene whose expression is epigenetically regulated by long non-coding RNAs (lncRNAs) within the locus. Furthermore, the long interspersed nuclear element-1 (LINE-1) L1MC1 transposon inserted in DPYD intronic transcript 1 (DPYD-IT1) lncRNA with its parasites, TcMAR-Tigger5b and pair of Alu repeats appears to be the “weakest link” within the DPYD gene liable to break. Identification of the precise mechanism through which DPYD is epigenetically regulated, and underlying reasons why exactly the break (FRA1E) happens, will consequently pave the way toward preventing severe toxicity to the antineoplastic drug 5-fluorouracil (5-FU) and development of the causative therapy for the dihydropyrimidine dehydrogenase deficiency.
... However, it has been shown that the presence of repetitive sequences in the 5 0untranslated region (5 0 -UTR) of some genes can influence expression. For example, repetitive elements in the 5 0 -UTR of a human zinc-finger gene modulate transcription and translation efficiency [36]. ...
The cytosolic sialidase Neu2 is known to be involved in myoblast differentiation. Here, we observed a Neu2 transcriptional induction during nerve growth factor, fibroblast growth factor 2 and epidermal growth factor treatments of PC12 cells, a favored model to study neuronal differentiation. The expression analysis of Neu2 deleted promoter revealed a remarkable increase of luciferase activity in treated PC12 cells, suggesting that in this cell line the Neu2 transcriptional levels are highly regulated.The enzymatic activity of cytosolic sialidase Neu2 was found to increase transiently only during differentiation, whereas was undetectable in untreated PC12 cells. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells.
... In some instances, TE insertion into intronic regions can cause mRNA destabilization, thereby reducing gene expression [45]. Similarly, some studies have suggested that TE insertion into the 5' or 3' region of a gene may alter its expression [46][47][48]. Thus, such a change in gene expression may, in turn, change the equilibrium of regulatory networks and result in disease conditions (reviewed in Konkel and Batzer [43]). ...
Approximately 45% of the human genome is comprised of transposable elements (TEs). Results from the Human Genome Project have emphasized the biological importance of TEs. Many studies have revealed that TEs are not simply "junk" DNA, but rather, they play various roles in processes, including genome evolution, gene expression regulation, genetic instability, and cancer disposition. The effects of TE insertion in the genome varies from negligible to disease conditions. For the past two decades, many studies have shown that TEs are the causative factors of various genetic disorders and cancer. TEs are a subject of interest worldwide, not only in terms of their clinical aspects but also in basic research, such as evolutionary tracking. Although active TEs contribute to genetic instability and disease states, non-long terminal repeat transposons are well studied, and their roles in these processes have been confirmed. In this review, we will give an overview of the importance of TEs in studying genome evolution and genetic instability, and we suggest that further in-depth studies on the mechanisms related to these phenomena will be useful for both evolutionary tracking and clinical diagnostics.
... Gene function can be affected by LINE-1 retrotransposition when the gene coding region is the target site of insertion. However, the ability of these retrotransposons to alter gene expression without interfering with the coding region was also documented (see Landry et al. 2001;Waterston et al. 2002). LINE-1 insertion in the UTR regions of a gene can affect the regulation of its transcription and translation. ...
Long interspersed nuclear elements-1 (LINE-1) are the most abundant and active retrotransposons in the mammalian genomes. Traditionally, the occurrence of LINE-1 sequences in the genome of mammals has been explained by the selfish DNA hypothesis. Nevertheless, recently, it has also been argued that these sequences could play important roles in these genomes, as in the regulation of gene expression, genome modelling and X-chromosome inactivation. The non-random chromosomal distribution is a striking feature of these retroelements that somehow reflects its functionality. In the present study, we have isolated and analysed a fraction of the open reading frame 2 (ORF2) LINE-1 sequence from three rodent species, Cricetus cricetus, Peromyscus eremicus and Praomys tullbergi. Physical mapping of the isolated sequences revealed an interspersed longitudinal AT pattern of distribution along all the chromosomes of the complement in the three genomes. A detailed analysis shows that these sequences are preferentially located in the euchromatic regions, although some signals could be detected in the heterochromatin. In addition, a coincidence between the location of imprinted gene regions (as Xist and Tsix gene regions) and the LINE-1 retroelements was also observed. According to these results, we propose an involvement of LINE-1 sequences in different genomic events as gene imprinting, X-chromosome inactivation and evolution of repetitive sequences located at the heterochromatic regions (e.g. satellite DNA sequences) of the rodents' genomes analysed.
... Table I summarizes the current state of knowledge concerning the ways in which retrotransposon may affect the structural and functional evolution of genes and genomes. (Shen et al., 1994) LTR-containing retrotransposons (Malik and Eickbush, 2001), and tRNA-derived SINEs (Ohshima et al., 1996) Recombination between REs may cause various diseases (Burwinkel and Kilimann, 1998; Kamp et al., 2000;Goodier and Kazazian Jr, 2008) SVA-mediated transduction duplicated the entire AMAC gene three times in the human genome (Xing et al., 2006) Mouse PMSE2b (Zaiss and Kloetzel, 1999) and PHGP pseudogenes (Boschan et al., 2002), TRIMCyp gene of owl monkey (Babushok et al., 2007) Formation of bipartite and tripartite chimeric elements in eukaryotic genomes (Fudal et al., 2005;Buzdin et al., 2007;Gogvadze et al., 2007) LTRs cause placental-specific expression of CYP19 (van de Lagemaat et al., 2003) and regulate transcription of the NAIP gene (Romanish et al., 2007); LTRs represent the only known promoter for the liver-specific BAAT gene (Carlton et al., 2003) Expression of salivary amylase in humans is a result of HERV-E integration (Meisler and Ting, 1993); ERV9 LTR are enhancer elements in the beta-globin locus control region (Long et al., 1998); Alu sequence is part of enhancer element of human CD8 alpha gene (Hambor et al., 1993) Muscle-specific inclusion of an Alu-derived exon in SEPN1 mRNA in humans (Lev-Maor et al., 2008); generation of alternative VEGFR-3 transcript due to the use of a non-canonical acceptor splice site within LTR sequence (Hughes, 2001) HERV-F LTR may function as an alternative polyadenylation site for gene ZNF195 (Kjellman et al., 1999) HERV-H LTRs are major polyadenylation signals for human HHLA2 and HHLA3 genes (Mager et al., 1999) RNAs transcribed from mouse B2 and human Alu SINEs have been found to control mRNA production at multiple levels (Ponicsan et al.2010 ) A part of Alu element is a transcriptional silencer of the human BRCA gene (Sharan et al., 1999); endogenous retroviral sequence RTVL-la may serve as silencer of the human Hpr gene (Maeda and Kim, 1990) Human-specific HERV-K LTRs generate antisense transcripts to SLC4A8 and IFT172 mRNAs B2 SINE element located in the murine growth hormone locus serves as a boundary to block the influence of repressive chromatin modification (Lunyak et al., 2007) drosophila LTR retrotranspson gypsy in the 5' region of the gene yellow blocks the action of the upstream located enhancers and is responsible for the pigmentation of cuticula (Dorsett, 1993) Alu and L1 segments in the 5'UTR of human ZNF177 gene modify gene expression on the protein level by decreasing translation efficiency (Landry et al., 2001) All three currently actively mobilizing non-LTR retrotransposon families-L1, SVA and Aluhave been identified as the causative agent of several genetic disorders (Konkel and Batzer, 2010) ...
Academic Dissertation To be presented for public criticism, with the permission of the Faculty of Biological and Environmental Sciences , University of Helsinki, in the auditorium 1041, Viikki Biocenter, Viikinkaari 5, Helsinki, on 9 December 2011, at 12 o'clock noon.
... Characterisation of the gene structure of the human B 1 R suggests that exon II is part of an Alu-J element that spans part of intron I, exon II and part of intron II [23]. Alu elements are small interspersed nucleotide elements which affect gene expression by influencing initiation of transcription and alternative splicing [47,48], initiation of translation, and translation efficiency [49][50][51][52]. More recently, the presence of Alu elements in exons and adjacent introns has been linked to forming circular RNAs, which have increasingly been reported as strong regulators of gene expression [53]. ...
The kinin B1 receptor (B1R) is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood.
We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV) of human B1R gene with a shorter 5′untranslated region. This novel SV is ≈35% less stable than the wild-type (WT) transcript in lung adenocarcinoma cells (H2126), but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE) −1020 to −766 bp in H2126; positive response element (PRE) −766 to −410 bp in 16HBE; −410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells) were found.
These findings reveal complex regulation of B1R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.
... Alternatively spliced Ex 21 of ATP8B1 is apparently an exonized Alu [27] element with the promoter P1 containing complementary Alu sequence. Alu sequences embedded in 59UTR have been shown to modulate both transcription and translation [28,29]. Other potent modulators of transcriptional and translational efficiency are Upstream Open Reading Frames (uORFs) which can affect gene expression by inhibition of mRNA stability and translational repression [30,31] . ...
Mutations in ATP8B1 gene were identified as a cause of low γ-glutamyltranspeptidase cholestasis with variable phenotype, ranging from Progressive Familial Intrahepatic Cholestasis to Benign Recurrent Intrahepatic Cholestasis. However, only the coding region of ATP8B1 has been described. The aim of this research was to explore the regulatory regions, promoter and 5'untranslated region, of the ATP8B1 gene.
5'Rapid Amplification of cDNA Ends using human liver and intestinal tissue was performed to identify the presence of 5' untranslated exons. Expression levels of ATP8B1 transcripts were determined by quantitative reverse-transcription PCR and compared with the non-variable part of ATP8B1. Three putative promoters were examined in vitro using a reporter gene assay and the main promoter was stimulated with chenodeoxycholic acid. Four novel untranslated exons located up to 71 kb upstream of the previously published exon 1 and twelve different splicing variants were found both in the liver and the intestine. Multiple transcription start sites were identified within exon -3 and the proximal promoter upstream of this transcription start site cluster was proven to be an essential regulatory element responsible for 70% of total ATP8B1 transcriptional activity. In vitro analysis demonstrated that the main promoter drives constitutive ATP8B1 gene expression independent of bile acids.
The structure of the ATP8B1 gene is complex and the previously published transcription start site is not significant. The basal expression of ATP8B1 is driven by a housekeeping-like promoter located 71 kb upstream of the first protein coding exon.
... Furthermore, L1 promoters contain binding sites for various transcription factors and regulatory proteins that can alter gene expression in response to various stimuli (45,81,82,124). L1 sequences can exert their influence on host gene expression by altering promoter specificity or strength (7,59,84,99,107). Not all TE interference is deleterious however, TE modification of gene expression may be responsible for genetic differences among species that translate into species-specific patterns of gene expression. ...
Historically the accumulated mass of mammalian transposable elements (TEs), particularly those located within gene boundaries, was viewed as a genetic burden potentially detrimental to the genomic landscape. This notion has been strengthened by the discovery that transposable sequences can alter the architecture of the transcriptome, not only through insertion, but also long after the integration process is completed. Insertions previously considered harmless are now known to impact the expression of host genes via modification of the transcript quality or quantity, transcriptional interference, or by the control of pathways that affect the mRNA life-cycle. Conversely, several examples of the evolutionary advantageous impact of TEs on the host gene structure that diversified the cellular transcriptome are reported. TE-induced changes in gene expression can be tissue- or disease-specific, raising the possibility that the impact of TE sequences may vary during development, among normal cell types, and between normal and disease-affected tissues. The understanding of the rules and abundance of TE-interference with gene expression is in its infancy, and its contribution to human disease and/or evolution remains largely unexplored.
... Alu repeats, which occupy more than one-tenth of the human genome, have been shown to harbor a large number of transcription factor binding sites (TFBSs)123, many of which have also been demonstrated to be functionally active. These have been mostly discovered during the course of characterization of regulatory sites in promoter regions of genes456789101112131415. Recently, genome wide informatics analyses have revealed substantial distribution of these sites in Alu repeats -for instance, nearly 90% of retinoic acid response element binding sites in human are in Alus [16]. ...
Alu RNAs are present at elevated levels in stress conditions and, consequently, Alu repeats are increasingly being associated with the physiological stress response. Alu repeats are known to harbor transcription factor binding sites that modulate RNA pol II transcription and Alu RNAs act as transcriptional co-repressors through pol II binding in the promoter regions of heat shock responsive genes. An observation of a putative heat shock factor (HSF) binding site in Alu led us to explore whether, through HSF binding, these elements could further contribute to the heat shock response repertoire.
Alu density was significantly enriched in transcripts that are down-regulated following heat shock recovery in HeLa cells. ChIP analysis confirmed HSF binding to a consensus motif exhibiting positional conservation across various Alu subfamilies, and reporter constructs demonstrated a sequence-specific two-fold induction of these sites in response to heat shock. These motifs were over-represented in the genic regions of down-regulated transcripts in antisense oriented Alus. Affymetrix Exon arrays detected antisense signals in a significant fraction of the down-regulated transcripts, 50% of which harbored HSF sites within 5 kb. siRNA knockdown of the selected antisense transcripts led to the over-expression, following heat shock, of their corresponding down-regulated transcripts. The antisense transcripts were significantly enriched in processes related to RNA pol III transcription and the TFIIIC complex.
We demonstrate a non-random presence of Alu repeats harboring HSF sites in heat shock responsive transcripts. This presence underlies an antisense-mediated mechanism that represents a novel component of Alu and HSF involvement in the heat shock response.
... Insertion of L1 elements in the antisense orientation can also truncate transcripts by creating new polyadenylation sites (a phenomenon known as gene breaking)[12]. Repetitive elements alter gene expression by introducing promoter regions near genes, or through many other documented mechanisms131415. It has been suggested that repetitive elements contribute to disease pathology by acting as superantigens or by causing an auto-immune response through molecular mimicry, but these hypotheses are largely speculative161718 . ...
Although nearly half of the human genome is comprised of repetitive sequences, the expression profile of these elements remains largely uncharacterized. Recently developed high throughput sequencing technologies provide us with a powerful new set of tools to study repeat elements. Hence, we performed whole transcriptome sequencing to investigate the expression of repetitive elements in human frontal cortex using postmortem tissue obtained from the Stanley Medical Research Institute.
We found a significant amount of reads from the human frontal cortex originate from repeat elements. We also noticed that Alu elements were expressed at levels higher than expected by random or background transcription. In contrast, L1 elements were expressed at lower than expected amounts.
Repetitive elements are expressed abundantly in the human brain. This expression pattern appears to be element specific and can not be explained by random or background transcription. These results demonstrate that our knowledge about repetitive elements is far from complete. Further characterization is required to determine the mechanism, the control, and the effects of repeat element expression.
... Además, se han identificado 7810 Alus intrónicas con posibilidad de ser exonizadas, lo que constituye un gran potencial para generar genes nuevos (Sorek et al., 2004). Otra vía de estudio tiene que ver con la regulación de la expresión génica, la cual se ve afectada a menudo por la presencia de TEs (Brosius, 1999; Hamdi et al., 2000; Nigumann et al., 2002 Landry et al., 2001). Además, en un trabajo reciente (Lagemaat et al., 2003) se demuestra que hay un exceso de TEs en los transcritos de genes con origen evolutivo reciente, mientras que se ven excluidos de los ARNm de genes con funciones básicas en el desarrollo o el metabolismo. ...
Tesis Univ. Granada. Departamento de Genética. Leída el 16 de septiembre de 2005
... Intronic insertions have also been associated with destabilization of the mRNA resulting in reduced expression [62]. In addition, insertions into the 5 and 3 prime region of genes can possibly alter their expression636465, reviewed in [66]. Alterations in gene expression increase the potential for altering equilibrium of regulatory networks, and thus augment susceptibility to certain diseases – including cancer. ...
It is now commonly agreed that the human genome is not the stable entity originally presumed. Deletions, duplications, inversions, and insertions are common, and contribute significantly to genomic structural variations (SVs). Their collective impact generates much of the inter-individual genomic diversity observed among humans. Not only do these variations change the structure of the genome; they may also have functional implications, e.g. altered gene expression. Some SVs have been identified as the cause of genetic disorders, including cancer predisposition. Cancer cells are notorious for their genomic instability, and often show genomic rearrangements at the microscopic and submicroscopic level to which transposable elements (TEs) contribute. Here, we review the role of TEs in genome instability, with particular focus on non-LTR retrotransposons. Currently, three non-LTR retrotransposon families - long interspersed element 1 (L1), SVA (short interspersed element (SINE-R), variable number of tandem repeats (VNTR), and Alu), and Alu (a SINE) elements - mobilize in the human genome, and cause genomic instability through both insertion- and post-insertion-based mutagenesis. Due to the abundance and high sequence identity of TEs, they frequently mislead the homologous recombination repair pathway into non-allelic homologous recombination, causing deletions, duplications, and inversions. While less comprehensively studied, non-LTR retrotransposon insertions and TE-mediated rearrangements are probably more common in cancer cells than in healthy tissue. This may be at least partially attributed to the commonly seen global hypomethylation as well as general epigenetic dysfunction of cancer cells. Where possible, we provide examples that impact cancer predisposition and/or development.
... Biologically, TE insertions into 5'-UTRs of transcripts have been shown to influence gene expression at the level of transcription through the creation of new transcription factor binding sites or by other transcriptional mecha- nisms363738. Alternatively, the presence of TE sequences could introduce deleterious uAUGs and uORFs to repress translational initiation394041 as we have demonstrated for selected T1 transcripts (Figure 7). ...
Retrotransposition is an important evolutionary force for the creation of new and potentially functional intronless genes which are collectively called retrogenes. Many retrogenes are expressed in the testis and the gene products have been shown to actively participate in spermatogenesis and other unique functions of the male germline. We have previously reported a cluster of retrogenes in the rat genome that encode putative TRAF- and POZ-domain proteins. Two of the genes, Rtdpoz-T1 and -T2 (abbreviated as T1 and T2), have further been shown to be expressed specifically in the rat testis.
We show here that the T1 and T2 genes are also expressed in the rat embryo up to days 16-17 of development when the genes are silenced until being re-activated in the adult testis. On database interrogation, we find that some T1/T2 exons are chromosomally duplicated as cassettes of 2 or 3 exons consistent with retro-duplication. The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR). Most importantly, the T1/T2 locus is embedded within a dense field of relics of transposable element (TE) derived mainly from LINE1 and ERV sequences, and the TE sequences are frequently exonised through alternative splicing to form the 5'-UTR sequences of the T1/T2 transcripts. In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence. Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed. We further demonstrate that the exonised 5'-UTR TE sequences could lead to the creation of upstream open reading frames resulting in translational repression.
Exonisation of TE sequences is a frequent event in the transcription of retrogenes during embryonic development and in the testis and may contribute to post-transcriptional regulation of expression of retrogenes.
... Previously ignored as junk, repetitive Alu elements are now known to modulate gene expression by alternate splicing, RNA editing and translation regulation [38][39][40] and may generate new enhancers, promoters and polyadenylation signals to many genes. Inverted alu are particularly unstable and are potential targets of RNA editing. ...
Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.
... Such events potentially generate genetic divergences that could drive evolution (Nekrutenko and Li, 2001). Additionally, since many transposons contain their own transcriptional regulatory elements, their mobilization can influence the expression patterns (White et al., 1994) or translational efficiency of neighboring genes (Landry et al., 2001). Finally, immunoglobulin and T cell receptor maturation require RAG1/RAG2-initiated V(D)J recombination. ...
Transposons populate the landscape of all eukaryotic genomes. Often considered purely genomic parasites, transposons can also benefit their hosts, playing roles in gene regulation and in genome organization and evolution. Peaceful coexistence with mobile elements depends upon adaptive control mechanisms, since unchecked transposon activity can impact long-term fitness and acutely reduce the fertility of progeny. Here, we review the conserved roles played by small RNAs in the adaptation of eukaryotes to coexist with their genomic colonists. An understanding of transposon-defense pathways has uncovered recurring themes in the mechanisms by which genomes distinguish "self" from "non-self" and selectively silence the latter.
... 14 As Alu elements are unique to primates, they might have played an important role in the evolution of primates through this way. Second, the full length or partial Alu elements have been shown to reside in the 5'UTRs of human growth hormone receptor (hGRH), 19 ZNF177, 20 and BRCA1. 21,22 These Alu elements can decrease mRNA translation efficiency, probably due to stable secondary structures formed by Alu that partially prevents translation initiation. ...
The Alu elements are conserved approximately 300 nucleotide long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Although the vast majority of Alu elements appear to be genetically inert, it has been tempting to consider the great majority of them as "junk DNA." However, a growing line of evidence suggests that transcribed Alu RNAs are in fact functionally involved in a number of diverse biological processes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to A-to-I editing by the ADAR enzymes. In this review we discuss the possible biological effects of Alu editing, with particular focus on the regulation of gene expression by inverted Alu repeats in the 3'-UTR regions of mRNAs.
... We found 25 ESTs that skip the Alu-containing exon (exon 26), whereas only three sequences (two of them were mRNAs) contained the exon (data not shown). The zinc finger gene ZNF177 has been reported to contain both an Alu and an L1 fragment in the constitutively spliced exon 4 (Baban et al. 1996; Landry et al. 2001 ). Apart from the two mR- NAs reported by (Baban et al. 1996), we failed to find a single EST that may be used to determine whether or not this exon is really constitutive. ...
Alu repetitive elements are found in approximately 1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.
... There are many different mechanisms that can account for the amplification and distribution of particularly successful coding or regulatory modules, but short replicative elements such as SINEs and MITEs would be particularly suitable candidates for such a function. SINEs are frequently found within or close to genes in Arabidopsis (Lenoir et al. 2001) and other organisms (Makalowski 1995), and it has been recently found that some of them can play an important biological role as coding or transcriptional regulatory regions (Shimamura et al. 1998;Ferrigno et al. 2001;Goodyer, Zheng, and Hendy 2001;Landry, Medstrand, and Mager 2001;Ackerman et al. 2002). Moreover, it has been proposed that B2 SINEs may have the potential to distribute a functional pol II promoter throughout the genome (Ferrigno et al. 2001). ...
Miniature inverted-repeat transposable elements (MITEs) are structurally similar to defective class II elements, but their
high copy number and the size and sequence conservation of most MITE families suggest that they can be amplified by a replicative
mechanism. Here we present a genome-wide analysis of the Emigrant family of MITEs from Arabidopsis thaliana. In order to be able to detect divergent ancient copies, and low copy number subfamilies with a different internal sequence
we have developed a computer program to look for Emigrant elements based solely on the terminal inverted-repeat sequence. We have detected 151 Emigrant elements of different subfamilies. Our results show that different bursts of amplification, probably of few active, or master,
elements, have occurred at different times during Arabidopsis evolution. The analysis of the insertion sites of the Emigrant elements shows that recently inserted Emigrant elements tend to be located far from open reading frames, whereas more ancient Emigrant subfamilies are preferentially found associated to genes.
... We found two independent cDNAs from the regeneration-competent blastema library that share homol-ogy to the OAX element. In addition, repetitive sequences in the untranslated regions (UTRs) of some genes may play a role in the regulation of transcriptional and translational efficiency, such as those found in the human zinc finger gene ZNF177 (Landry et al., 2001) and the Hlx homeobox gene required for hepatic growth and development (Bates et al., 2001). ...
Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.
... Mounting evidence suggests that cis-acting elements in untranslated regions can play a significant role in enhancing the transcription of some genes. This has been reported in several studies, including the human hematopoietic prostaglandin D synthase gene, in which the untranslated first exon showed 60% of the activity of the entire 1044/290 region (Fujimori et al., 2000; Hiroi et al., 2001; Landry et al., 2001; Wang et al., 2002; Cheng et al., 2003). ...
Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating oxygen-dependent gene expression and is involved in a range of pathways implicated in cellular survival, proliferation, and development. While the posttranslational regulation of HIF-1alpha is well characterized, the relative importance of its control at the transcriptional level during development remains less clear. Although the mouse and human promoter regions have been analyzed in vitro, to date, there has been no in vivo analysis of any vertebrate HIF-1alpha promoter. To investigate the transcriptional regulation of HIF-1alpha during development of the amphibian Xenopus laevis, we have described the gene's expression pattern and isolated the xHIF-1alpha upstream regulatory regions. We show xHIF-1alpha mRNA to be constitutively expressed at low levels throughout embryogenesis, but with significant up-regulation during gastrula stages, and subsequently, in specific regions of the central nervous system and axial tissues. Our functional analysis using a series of truncated xHIF-1alpha promoter constructs demonstrates that a 173-bp region of the proximal promoter, which is 100% conserved among five allelic variants, is sufficient to drive correct expression in transgenic embryos. Although these results are corroborated by a parallel set of in vitro transfection experiments in a Xenopus cell line, some key differences suggest the importance of using transgenic methods in conjunction with in vitro assays.
... It is not necessary to target the coding region to affect gene function and the ability of retroelements to alter gene regulation without interfering with the peptide sequence itself is well-documented. We recently demonstrated that retroelements within the 5) UTR of a zinc-finger gene affect gene regulation ( Landry et al., 2001). A possible constitutive recruitment of a retroelement exon into gene transcripts is represented by the human kinin B1 receptor (Cayla et al., 2002) which incorporates an Alu element in the 5) UTR of the gene transcript, suggesting that TEs are used to fine tune gene expression and regulation. ...
Transposable elements (TEs) are present in all organisms and nearly half of the human and mouse genome is derived from ancient transpositions. This fact alone suggests that TEs have played a major role in genome organization and evolution. Studies undertaken over the last two decades or so clearly show that TEs of various kinds have played an important role in organism evolution. Here we review the impact TEs have on the evolution of gene regulation and gene function with an emphasis on humans. Understanding the mechanisms resulting in genomic change is central to our understanding of gene regulation, genetic disease and genome evolution. Full comprehension of these biological processes is not possible without an in depth knowledge of how TEs impact upon the genome.
... By 1997, Britten was able to find more than 20 examples that conformed to all four of these criteria and many more similar examples have been uncovered since that time. For instance, a number of cases where human TEs can be shown to serve as promoters for adjacent genes have recently been identified (Landry et al., 2001Landry et al., , 2002 Medstrand et al., 2001; Dunn et al., 2003). The most extensive literature survey to date of TE contributions to host gene regulation identified almost 80 cases where regulatory elements of vertebrate genes are derived from TEs (Brosius, 1999). ...
The evolutionary implications of transposable element (TE) influences on gene regulation are explored here. An historical perspective is presented to underscore the importance of TE influences on gene regulation with respect to both the discovery of TEs and the early conceptualization of their potential impact on host genome evolution. Evidence that points to a role for TEs in host gene regulation is reviewed, and comparisons between genome sequences are used to demonstrate the fact that TEs are particularly lineage-specific components of their host genomes. Consistent with these two properties of TEs, regulatory effects and evolutionary specificity, human-mouse genome wide sequence comparisons reveal that the regulatory sequences that are contributed by TEs are exceptionally lineage specific. This suggests a particular mechanism by which TEs may drive the diversification of gene regulation between evolutionary lineages.
... Although the significance of the long 5 0 UTR of A2 cDNA remains unclear, it may affect the translation of the A2 chain mRNA, because in the purification of the lethal toxin from the venom of B. flaviceps we were unsuccessful in detecting the toxin that possesses the amino acid sequence of the A2 chain. Recently, it was shown that retroelement sequences within the 5 0 UTR of mRNAs could repress translation (Landry et al., 2001). In addition, the efficiency of transcript translation has been shown to depend on the structure of the 5 0 UTR. ...
The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.
Discovered by their capacity to mobilize across the genome by the Nobel laureate Barbara McClintock while studying plant cytogenetics around the middle of the past century, transposable elements (TEs) are becoming the focus of intense research due to their capacity to regulate gene expression and shape individuals’ genomes. This chapter introduces the reader into the nomenclature, structure, and classification of eukaryotic TEs and reviews the influence of TEs on the acquisition of new gene functions, some of them essential for the adaptation of organisms to environmental stress. It also provides mechanistic details for the regulation of TEs and the connections of these elements with the genetic and epigenetic cell regulatory networks. Examples are provided to illustrate the relevance of TEs in the biology of eukaryotes and the participation of TEs in human disease.
A consensus nucleotide sequence of long terminal repeats (LTRs) of endogenous human-specific retroviruses of the K family (HERV-K) was constructed and used for the genome-wide search for homologies in international databases. There were revealed 142 LTRs, 12 of which were localized in introns of unique human genes. It was found for the first time that ten intron LTRs are absent in the orthologic loci of the chimpanzee genome and the orientation of nine of them is opposite to the transcription direction of the corresponding human genes. A hypothesis was propounded that the found LTRs affect the gene expression by initiation of the antisense RNA synthesis.
The polarized memory effect (the memory in which switching between ON
and OFF states depends on the polarity of the bias voltage) on the
Se-SnO2 system is described. This effect is observed on the
samples with Se films 300˜5000 Å in thickness. The ratio of
the resistance of OFF state ROFF to that of ON state
RON is about three orders of magnitude. In the case of the
film with thickness 103 Å, the threshold voltage from
OFF to ON states Vth is 15 V, and the reverse voltage from ON
to OFF states Vr is 3.5 V.
Auxin plays crucial roles in plant development. Auxin-binding protein 1 (ABP1) is an auxin receptor required to coordinate cell division and expansion during postembryonic shoot development, and differential auxin responses during root growth. While ABP1 is encoded by a single gene in maize, multiple gene copies exist in teosinte, the wild relatives of maize. We have previously shown that some of the differences between these genes are caused by multiple transposon insertions in the promoter. Here we show that the different ABP1 promoter types confer differential gene expression levels on a firefly luciferase reporter gene. We also discovered a negative regulatory sequence upstream of the conserved transcriptional start site. When this sequence is insulated by an Ac-like transposon or deleted in natural ABP1 gene variants, expression levels are enhanced. Promoters combining both a MITE and a solo-LTR showed small but significant increase in expression compared to those containing only one insertion. This increase seems to be additive, suggesting that it may be due to enhancer sequences present within these transposons. Our results point to a potential role of the ABP1-associated transposons in the modulation of ABP1 gene expression.
Several classes of small noncoding RNAs are key players in cellular metabolism including mRNA decoding, RNA processing, and mRNA stability. Here we show that a tRNA(Asp) isodecoder, corresponding to a human tRNA-derived sequence, binds to an embedded Alu RNA element contained in the 3' UTR of the human aspartyl-tRNA synthetase mRNA. This interaction between two well-known classes of RNA molecules, tRNA and Alu RNA, is driven by an unexpected structural motif and induces a global rearrangement of the 3' UTR. Besides, this 3' UTR contains two functional polyadenylation signals. We propose a model where the tRNA/Alu interaction would modulate the accessibility of the two alternative polyadenylation sites and regulate the stability of the mRNA. This unique regulation mechanism would link gene expression to RNA polymerase III transcription and may have implications in a primate-specific signal pathway.
Transposable elements (TEs) are increasingly being recognized as powerful facilitators of evolution. We propose the TE-Thrust hypothesis to encompass TE-facilitated processes by which genomes self-engineer coding, regulatory, karyotypic or other genetic changes. Although TEs are occasionally harmful to some individuals, genomic dynamism caused by TEs can be very beneficial to lineages. This can result in differential survival and differential fecundity of lineages. Lineages with an abundant and suitable repertoire of TEs have enhanced evolutionary potential and, if all else is equal, tend to be fecund, resulting in species-rich adaptive radiations, and/or they tend to undergo major evolutionary transitions. Many other mechanisms of genomic change are also important in evolution, and whether the evolutionary potential of TE-Thrust is realized is heavily dependent on environmental and ecological factors. The large contribution of TEs to evolutionary innovation is particularly well documented in the primate lineage. In this paper, we review numerous cases of beneficial TE-caused modifications to the genomes of higher primates, which strongly support our TE-Thrust hypothesis.
Comparison of a full collection of the transposable element (TE) sequences of vertebrates with genome sequences shows that the human genome makes 655 perfect full-length matches. The cause is that the human genome contains many active TEs that have caused TE inserts in relatively recent times. These TE inserts in the human genome are several types of young Alus (AluYa5, AluYb8, AluYc1, etc.). Work in many laboratories has shown that such inserts have many effects including changes in gene expression, increases in recombination, and unequal crossover. The time of these very effective changes in the human lineage genome extends back about 4 million years according to these data and very likely much earlier. Rapid human lineage-specific evolution, including brain size is known to have also occurred in the last few million years. Alu insertions likely underlie rapid human lineage evolution. They are known to have many effects. Examples are listed in which TE sequences have influenced human-specific genes. The proposed model is that the many TE insertions created many potentially effective changes and those selected were responsible for a part of the striking human lineage evolution. The combination of the results of these events that were selected during human lineage evolution was apparently effective in producing a successful and rapidly evolving species.
The traditional understanding that proteins are the only effectors of gene function has been challenged by the discovery of a group of genes that do not encode proteins (non-coding genes [ncGs]). The role of ncGs in the pathogenesis and potentially the treatment of several human diseases is increasingly being confirmed. A robust collection of literature exists to support the theory of the involvement of ncGs and their non-coding RNA (ncRNA) transcripts in the pathogenesis of cancer. This review focuses on the role of ncRNAs in human carcinogenesis and describes why deciphering the function of these RNAs might lead to the development of new anticancer drugs.
Transposable elements (TEs) are powerful facilitators of genome evolution, and hence of phenotypic diversity as they can cause genetic changes of great magnitude and variety. TEs are ubiquitous and extremely ancient, and although harmful to some individuals, they can be very beneficial to lineages. TEs can build, sculpt, and reformat genomes by both active and passive means. Lineages with active TEs or with abundant homogeneous inactive populations of TEs that can act passively by causing ectopic recombination are potentially fecund, adaptable, and taxonate readily. Conversely, taxa deficient in TEs or possessing heterogeneous populations of inactive TEs may be well adapted in their niche, but tend to prolonged stasis and may risk extinction by lacking the capacity to adapt to change, or diversify. Because of recurring intermittent waves of TE infestation, available data indicate a compatibility with punctuated equilibrium, in keeping with widely accepted interpretations of evidence from the fossil record. We propose a general and holistic synthesis on how the presence of TEs within genomes makes them flexible and dynamic, so that genomes themselves are powerful facilitators of their own evolution.
Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic 'junk'.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a multi-author review).
The wide distribution of GnRH-II and conservation of its structure over all vertebrate classes suggest that the neuropeptide possesses vital biological functions. Although recent studies have shown that the expression of the human GnRH-II gene is regulated by cAMP and estrogen, the molecular mechanisms governing its basal transcription remain poorly understood. Using the neuronal TE-671 and placental JEG-3 cells, we showed that the minimal human GnRH-II promoter was located between nucleotide -1124 and -750 (relative to the translation start codon) and that the untranslated exon 1 was important to produce full promoter activity. Two putative E-box binding sites and one Ets-like element were identified within the first exon, and mutational analysis demonstrated that these cis-acting elements functioned cooperatively to stimulate the human GnRH-II gene transcription. EMSAs, UV cross-linking, and Southwestern blot analyses indicated that the basic helix-loop-helix transcription factor AP-4 bound specifically to the two E-box binding sites, whereas an unidentified protein bound to the Ets-like element. The functional importance of AP-4 in controlling human GnRH-II gene transcription was demonstrated by overexpression of sense and antisense full-length AP-4 cDNAs. Taken together, our present data demonstrate a novel mechanism in stimulating basal human GnRH-II gene transcription mediated by cooperative actions of multiple regulatory elements within the untranslated first exon of the gene.
Cathepsin B is a papain-family cysteine protease that is normally located in lysosomes, where it is involved in the turnover of proteins and plays various roles in maintaining the normal metabolism of cells. This protease has been implicated in pathological conditions, e.g., tumor progression and arthritis. In disease conditions, increases in the expression of cathepsin B occur at both the gene and protein levels. At the gene level, the altered expression results from gene amplification, elevated transcription, use of alternative promoters and alternative splicing. These molecular changes lead to increased cathepsin B protein levels and in turn redistribution, secretion and increased activity. Here we focus on the molecular regulation of cathepsin B and attendant implications for tumor progression and arthritis. The potential of cathepsin B as a therapeutic target is also discussed.
The cytosolic sialidase Neu2 is known to be involved in myoblast differentiation. Here, we observed a Neu2 transcriptional induction during nerve growth factor, fibroblast growth factor 2 and epidermal growth factor treatments of PC12 cells, a favored model to study neuronal differentiation. The expression analysis of Neu2 deleted promoter revealed a remarkable increase of luciferase activity in treated PC12 cells, suggesting that in this cell line the Neu2 transcriptional levels are highly regulated. The enzymatic activity of cytosolic sialidase Neu2 was found to increase transiently only during differentiation, whereas was undetectable in untreated PC12 cells. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells.
It is very likely that formation of new genes is the main pathway of molecular evolution in living organisms. Many such genes are products of preexisting reshuffling of genetic material. In these processes a very important role is played by mutations associated with the activity of transposable elements, mostly retroelements (REs) for higher eukaryotes. The life cycle of REs involves a stage of so-called reverse transcription of their RNA intermediates, i.e. synthesis of complementary DNA on an RNA template. Transcriptionally active sequences of RE origin are referred to as retrogenes. REs create chimeric genes by a variety of mechanisms: new RE insertions, recombinations between RE sequences, formation of functional gene active pseudogenes and template switches during reverse transcription of messenger RNA. The abovementioned events are also able to give rise to new RE families. These mechanisms are reviewed here along with the description of major RE groups.
Autonomous non-long terminal repeat retrotransposons are commonly referred to as long interspersed elements (LINEs). Short non-autonomous elements that borrow the LINE machinery are called SINES. The Entamoeba histolytica genome contains three classes of LINEs and SINEs. Together the EhLINEs/SINEs account for about 6% of the genome. The recognizable functional domains in all three EhLINEs included reverse transcriptase and endonuclease. A novel feature was the presence of two types of members-some with a single long ORF (less frequent) and some with two ORFs (more frequent) in both EhLINE1 and 2. The two ORFs were generated by conserved changes leading to stop codon. Computational analysis of the immediate flanking sequences for each element showed that they inserted in AT-rich sequences, with a preponderance of Ts in the upstream site. The elements were very frequently located close to protein-coding genes and other EhLINEs/SINEs. The possible influence of these elements on expression of neighboring genes needs to be determined.
AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25–26
and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate
transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353–613). We
named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent
cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50
kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These
findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of
AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor
cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, γ-irradiation treatment provokes AIFsh up-regulation.
Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent
cell death in tumor formation/suppression.
The expression of imprinted genes is mediated by allele-specific epigenetic modification of genomic DNA and chromatin, including parent of origin-specific DNA methylation. Dysregulation of these genes causes a range of disorders affecting pre- and post-natal growth and neurological function. We investigated a cohort of 12 patients with transient neonatal diabetes whose disease was caused by loss of maternal methylation at the TNDM locus. We found that six of these patients showed a spectrum of methylation loss, mosaic with respect to the extent of the methylation loss, the tissues affected and the genetic loci involved. Five maternally methylated loci were affected, while one maternally methylated and two paternally methylated loci were spared. These patients had higher birth weight and were more phenotypically diverse than other TNDM patients with different aetiologies, presumably reflecting the influence of dysregulation of multiple imprinted genes. We propose the existence of a maternal hypomethylation syndrome, and therefore suggest that any patient with methylation loss at one maternally-methylated locus may also manifest methylation loss at other loci, potentially complicating or even confounding the clinical presentation.
The genomes of all eukaryotes contain multiple copies of DNA sequences that are related to sequences found in infectious retroviruses (for review, see Coffin, 1984; Garfinkel, 1992). These elements are transmitted through the germ line as stable Mendelian genes, yet they exhibit structural and sequence similarities to infectious exogenous retroviruses. It is these similarities that have led investigators to speculate that endogenous retroviruses are remnants of prior infections with exogenous retroviral agents and, with evolutionary time, changes have occurred to make them no longer infectious or pathogenic. These speculations have been supported with experimental studies that show that the genomes of infectious, exogenous retroviruses can integrate into the host chromosome and be inherited through the germ line. Since retroviruses are thought to have evolved from retrotransposons (Temin, 1980, 1992), it is also possible that some endogenous retrovirus-related sequences are actually precursors of infectious forms. In either case, once they are part of the host genome, these proviruses can serve as a pool of genetic material that exogenous viruses can use to produce variants with altered host specificities and phenotypes; they can encode gene products that compete for or complement in trans retrovirus function(s); and they can, themselves, act as insertional mutagens to change the regulation of host genes.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution.
Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human
genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
The 5′ and 3′ untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling
mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/Biowww/#UTRdb), a specialized database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are
enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns
already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the
UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore,
UTRdb entries have been annotated for the presence of repetitive elements.
The important role the untranslated regions of eukaryotic mRNAs may play in gene regulation and expression is now widely acknowledged.
For this reason we developed UTRdb, a specialized database of 5′- and 3′-untranslated sequences of eukaryotic mRNAs cleaned
from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases, including the
presence of functional patterns already demonstrated by experimental analysis to have some functional role. A collection of
such patterns is being collected in UTRsite database (http://bio-www.ba.cnr.it:8000/srs5/) which can also be used with appropriate computational tools to detect known functional patterns contained in mRNA untranslated
regions.
The polymerase chain reaction was used to detect expression of retroviral sequences with oligonucleotide primers derived from conserved regions of the retroviral genome. Four primer pairs derived from gag and one from pol were used in amplification of reverse-transcribed total RNA prepared from peripheral blood mononuclear cells of seven blood donors. The amplification pattern was the same from each of the seven samples. Sequencing of cloned amplification products revealed that at least three subclasses of sequences related to the human endogenous retroviruses (HERV) RTVL-H, HERV-E and HERV-K, are expressed in peripheral blood mononuclear cells of healthy individuals. This has not been previously reported.
Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.
In the simple adenovirus 2 early 1B (E1B) promoter, moving the single Sp1 transcription factor binding site (GC box) from its wild-type position eight base pairs (bp) from the TATA box to 30 bp upstream is equivalent to its deletion (Wu, L., and Berk, A. (1988) Genes & Dev. 2, 403-411). In more complex promoters, multiple upstream elements regulate transcription over greater distances. To understand these spacing constraints, we placed two GC boxes in phase at various distances from the E1B TATA box. Whereas one GC box at eight bp from TATA increases transcription in vivo 5-fold compared with TATA alone (Wu, L., Rosser, D. S. E., Schmidt, M. C., and Berk, A. (1987) Nature 326, 512-515), two GC boxes increased in vivo transcription 25-30-fold. Transcriptional stimulation by two GC boxes fell off rapidly in vivo at 30 bp from TATA, remained approximately constant through 50 bp, then decreased again at 70 bp. Consequently, two GC boxes have a multiplicative effect at eight bp from TATA and continue to stimulate transcription at a greater distance from TATA than a single Sp1 site. Quantitatively different results were observed for in vitro transcription using a nuclear extract; a more linear fall off with increasing distance from TATA was observed. Separating the two GC boxes progressively decreased transcription. E1A stimulation of these promoters in vivo indicates that Sp1 transcription control is independent of E1A transactivation.
By a simple direct blot hybridization strategy, the existence of human Alu family subfamilies is confirmed. Using consensus
restriction cleavage sites, individual bands can be resolved from genomic human DNA digests corresponding to three distinct
Alu subfamilies. Digestion with methylation sensitive and insensitive restriction enzymes shows that the numerous CpG residues
in the youngest Alu subfamilies are largely methylated in vivo, suggesting a model for the transcriptional regulation of Alu repeats.
Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts--encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins--that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation.
The biochemical analysis of cellular trans-activators involved in promoter recognition provides an important step toward understanding the mechanisms of gene expression in animal cells. The promoter selective transcription factor, Sp1, has been purified from human cells to more than 95 percent homogeneity by sequence-specific DNA affinity chromatography. Isolation and renaturation of proteins purified from sodium dodecyl sulfate polyacrylamide gels allowed the identification of two polypeptides (105 and 95 kilodaltons) as those responsible for recognizing and interacting specifically with the GC-box promoter elements characteristic of Sp1 binding sites.
The rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated under rare conditions. One circumstance that has been suggested to allow dual initiation is close apposition of a second AUG codon. A possible mechanism might be that the scanning 40S ribosomal subunit flutters back and forth instead of stopping cleanly at the first AUG. This hypothesis seems to be ruled out by evidence presented herein that in certain mRNAs, the first of two close AUG codons is recognized uniquely. To achieve this, the 5' proximal AUG has to be provided with the full consensus sequence; even small departures allow a second nearby AUG codon to be reached by leaky scanning. This context-dependent leaky scanning unexpectedly fails when the second AUG codon is moved some distance from the first. A likely explanation, based on analyzing the accessibility of a far-downstream AUG codon under conditions of initiation versus elongation, is that 80S elongating ribosomes advancing from the 5' proximal start site can mask potential downstream start sites.
Dispersion of repetitive sequence elements is a source of genetic variability that contributes to genome evolution. Alu elements, the most common dispersed repeats in the human genome, can cause genetic diseases by several mechanisms, including de novo Alu insertions and splicing of intragenic Alu elements into mRNA. Such mutations might contribute positively to protein evolution if they are advantageous or neutral. To test this hypothesis, we searched the literature and sequence databases for examples of protein-coding regions that contain Alu sequences: 17 Alu 'cassettes' inserted within 15 different coding sequences were found. In three instances, these events caused genetic diseases; the possible functional significance of the other Alu-containing mRNAs is discussed. Our analysis suggests that splice-mediated insertion of intronic elements is the major mechanism by which Alu segments are introduced into mRNAs.
Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation
state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical
and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues
such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the
paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear
to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG
dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state
of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.
Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
TRANSFAC, TRRD (Transcription Regulatory Region Database) and COMPEL are databases which store information about transcriptional
regulation in eukaryotic cells. The three databases provide distinct views on the components involved in transcription: transcription
factors and their binding sites and binding profiles (TRANSFAC), the regulatory hierarchy of whole genes (TRRD), and the structural
and functional properties of composite elements (COMPEL). The quantitative and qualitative changes of all three databases
and connected programs are described. The databases are accessible via WWW:http://transfac.gbf.de/TRANSFAC or http://www.bionet.nsc.ru/TRRD
Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level
of these transcripts is sensitive to the translational state of the cell. To determine whether Alu RNA functions in translational
homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR. We found that overexpression
of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory
effect on PKR. Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation
both in vitro and in vivo. Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas
transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo. Cycloheximide treatment of cells decreased
PKR activity, coincident with an increase in Alu RNA. These observations suggest that the increased levels of Alu RNAs caused
by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation. This provides a functional
role for mammalian short interspersed elements, prototypical junk DNA.
We studied the Hga I polymorphism (46 C/T) in the 5'-untranslated region of the coagulation factor XII (FXII) gene corresponding to four bases upstream from the ATG translation initiation codon. By using allele-specific restriction analysis with restriction endonuclease Hga I, the allele frequency of 46C/T was estimated to be 0.27/0.73 in Orientals (allele number =152), and conversely, 0.8/0.2 in Caucasians (allele number =40). Because it has been reported that plasma levels of FXII were lower in Orientals than in Caucasians, we investigated the relationship between this polymorphism and plasma levels of FXII. As a result, there were significant differences in plasma FXII levels between these three allele types: C/C,170+/-38% (178+/-27%); C/T, 141+/-29% (123+/-34%); and T/T, 82+/-19% (61+/-11%) [FXII activity (FXII antigen levels)]. In heterozygotes of 46 C/T both alleles were equally transcribed in hepatocytes, as determined by reverse transcription polymerase chain reaction (RT-PCR), suggesting little influence of the polymorphism at the level of transcription or on the stability of mRNA. In in vitro transcription/translation analysis, less FXII was produced from cDNA containing 46 T than from that containing 46 C. Therefore, it is highly likely that the 46 T polymorphism in the FXII gene decreased the translation efficiency and led to low plasma levels of FXII activity and antigen, probably due to the creation of another ATG codon and/or impairment of the consensus sequence for the translation initiation scanning model.
The 'master' human mobile element, the L1 retrotransposon, has come of age as a biological entity. Knowledge of how it retrotransposes in vivo, how its proteins act to retrotranspose other poly A elements and the extent of its role in shaping the human genome should emerge rapidly over the next few years. We review the impact of retrotransposons and how new insight is likely to lead to important practical applications for these intriguing mobile elements.
An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.
While the significance of middle repetitive elements had been neglected for a long time, there are again tendencies to ascribe most members of a given middle repetitive sequence family a functional role--as if the discussion of SINE (short interspersed repetitive elements) function only can occupy extreme positions. In this article, I argue that differences between the various classes of retrosequences concern mainly their copy numbers. Consequently, the function of SINEs should be viewed as pragmatic such as, for example, mRNA-derived retrosequences, without underestimating the impact of retroposition for generation of novel protein coding genes or parts thereof (exon shuffling by retroposition) and in particular of SINEs (and retroelements) in modulating genes and their expression. Rapid genomic change by accumulating retrosequences may even facilitate speciation [McDonald, J.F., 1995. Transposable elements: possible catalysts of organismic evolution. Trends Ecol. Evol. 10, 123-126.] In addition to providing mobile regulatory elements, small RNA-derived retrosequences including SINEs can, in analogy to mRNA-derived retrosequences, also give rise to novel small RNA genes. Perhaps not representative for all SINE/master gene relationships, we gained significant knowledge by studying the small neuronal non-messenger RNAs, namely BC1 RNA in rodents and BC200 RNA in primates. BC1 is the first identified master gene generating a subclass of ID repetitive elements, and BC200 is the only known Alu element (monomeric) that was exapted as a novel small RNA encoding gene.
The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
We studied the Hga I polymorphism (46 C/T) in the 5′-untranslated region of the coagulation factor XII (FXII) gene corresponding to four bases upstream from the ATG translation initiation codon. By using allele-specific restriction analysis with restriction endonuclease Hga I, the allele frequency of 46C/T was estimated to be 0.27/0.73 in Orientals (allele number =152), and conversely, 0.8/0.2 in Caucasians (allele number =40). Because it has been reported that plasma levels of FXII were lower in Orientals than in Caucasians, we investigated the relationship between this polymorphism and plasma levels of FXII. As a result, there were significant differences in plasma FXII levels between these three allele types: C/C,170±38% (178±27%); C/T, 141±29% (123±34%); and T/T, 82±19% (61±11%) [FXII activity (FXII antigen levels)]. In heterozygotes of 46 C/T both alleles were equally transcribed in hepatocytes, as determined by reverse transcription polymerase chain reaction (RT-PCR), suggesting little influence of the polymorphism at the level of transcription or on the stability of mRNA. In in vitro transcription/translation analysis, less FXII was produced from cDNA containing 46 T than from that containing 46 C. Therefore, it is highly likely that the 46 T polymorphism in the FXII gene decreased the translation efficiency and led to low plasma levels of FXII activity and antigen, probably due to the creation of another ATG codon and/or impairment of the consensus sequence for the translation initiation scanning model.
The RNA polymerase II transcription factor Sp l is a protein that binds to specific D NA sequences and activates RNA synthesis from a select group of promoters. Spl and related factors appear to be important for modulation of gene expression in higher organisms. Control of the rate of transcription initia-tion is one means by which the expres-sion of genes can be varied. Regulation of transcription initiation is well charac-terized in prokaryotes such as Escher-ichia coli, but in higher organisms, such as humans, this phenomenon is only beginning to be clarified. One common approach to this problem has been the identification of important c/s-acting DNA sequences in the region surround-ing transcription initiation sites. For synthesis of mRNA by RNA polymerase II, these studies have revealed some im-portant and distinct promoter elements, such as specific sequences within 100 nucleotides upstream of the start site that contribute to the efficiency of mRNA synthesis, and an AT-rich region of DNA (25-30 bp upstream of the RNA start site and sometimes called the TATA box) that appears to fix the site of transcription initiation 1-6. Eukaryotic transcription can also be greatly influ-enced by control elements known as enhancers, which can increase the level of transcription from long distances (at least 2 kb) and from both orientations, when either upstream or downstream of the RNA start site 3,7-H. Promoter mapping studies of several eukaryotic genes in vivo and in vitro have revealed the importance of a GGGCGG hexanucleotide (GC box) 3,4,12,13 and a CCAAT sequence (CAAT box) 14, which are often found 40-100 nucleo-tides upstream of the start site of trans-cription. These elements appear to play a critical role in directing efficient trans-cription from a select class of mammalian promoters. Examples include: the Simian Virus 40 early promoter3,4,n,13, which contains six tandemly arranged GC boxes; the mammalian I]-globin promoters 15-17, which each possess a single CAAT box; and the herpes simplex virus (HSV) thymidine kinase (TK) promoterS,6,18,w, which has two GC boxes that flank a single CAAT box. To understand how the GC and CAAT boxes affect the level of transcription in the cell, it is necessary to complement the genetic mapping experiments with biochemical identification and purifica-tion of factors that interact specifically with GC and CAAT boxes to modulate the synthesis of RNA. By characteriza-tion of such factors, it should be possible to elucidate the mechanisms of prom-otor-specific variation of transcription. Two transcription factors, termed Spl and CTF (CAAT-binding transcription factor), have been isolated recently from mammalian cells 19-21. Spl and CTF bind to GC and CAAT boxes, respec-tively 19,22 (and S. McKnight, pers. com-mun.). Because Spl has been studied more extensively than CTF, we focus, in this review, mainly on the promoter-specific activation of transcription by Spl. Properties of Spl
To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.
We have cloned and sequenced a cDNA copy of in vitro-polyadenylated 7SL RNA of HeLa cells. The cloned fragment is 303 bp long and has a composite structure. A central block of 140 bp is homologous to a new set of human middle-repetitive sequences. This block appears to be inserted in an Alu consensus sequence, 100 bp from the 5' end and 40 bp from the 3' end of the Alu monomer. Two 6 bp direct repeats are found at the junction between the Alu flanking sequences and the central element. The analysis of several clones shows the existence of sequence microheterogeneity in the 5' portion of the molecule. The 7L DNA probably represents a subset of the Alu family of DNA, highly conserved in evolution.
The Alu repeat sequence is estimated to account for 5% of human genomic DNA. The precise relationship of Alu sequences to human fully spliced cDNA has yet to be determined, although many new protocols for cloning cDNAs either depend on the presence of Alus or--more usually--rely on their absence in a population of messages. Previous estimates of the percentage of fully spliced human transcripts that contain Alu repeats have relied on hybridization procedures. Here we have gone directly to the DNA sequence by extracting over 1600 entries from GenBank that are described as human complete cDNAs, and we have assessed the frequency with which the Alu repeat sequence occurs in these sequences. We find that 5% of fully spliced human cDNAs contain Alu sequences. In addition, we have quantified the appearance of Alus in the different cDNA regions, 5' untranslated region (UTR), coding region, and 3' UTR. The vast majority of Alus are found in the 3' UTR, but 14% lie in the 5' UTR, and very rarely an Alu sequence is present within, or partially within, the coding region of the transcript.
Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.
During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5' termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C2H2 type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5' putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types.
The human blood platelet fibrinogen receptor, integrin alphaIIbbeta3 (glycoprotein IIb-IIIa) is an archetypal member of the integrin family of adhesive molecules and is the only integrin encoded by genes physically linked in the genome. Because studies on the normal and abnormal expression of any gene require a thorough understanding of its organization, the initial goals of the current study were to determine the size and complete the genomic organization for the beta3 gene. We now report the isolation of the entire beta3 gene in a single P1 plasmid and for the first time have linked the first and second exons on a contiguous fragment of DNA. Using pulsed-field gel analysis, we determined the full size of the beta3 gene to be 63 kb and show a large (16.7 kb) first intron; based on this information, we propose a uniform numbering system for the beta3 exons. We have completed the 5' genomic structure and generated a long-range restriction map. The promoter and the 5' end of the first intron were found to have approximately 50% sequence identity with a region of the avian beta3 gene known to possess functional transcriptional activity. Analysis of three different homologous regions led to the identification of a sequence in the 5'-UTR of the human gene, CCGCGGGAGG, which shares 90% identity with the avian gene and which bound nuclear proteins in DNaseI and electrophoretic mobility shift assay studies. Mutating this sequence caused a 2.6-fold reduction in reporter gene activity. In these studies we have (1) determined the full length and 5' organization of the beta3 gene, (2) identified a large region of homology between the 5' regions of the avian and human genes, and (3) identified a sequence in the 5'-UTR that augments gene expression. Knowing the genomic structure of beta3 has permitted the uncovering of new mechanisms of mutagenesis causing Glanzmann thrombasthenia (Jin et al, J Clin Invest 98:1745, 1996), and our findings will be valuable for such genetic analyses as well as for studies on the transcriptional regulation of beta3 and other integrin genes.
DNA methylation has an essential regulatory function in mammalian development, serving to repress nontranscribed genes stably in differentiated adult somatic cells. Recent data implicate transcriptional repressors specific for methylated DNA and chromatin assembly in this global control of gene activity. The assembly of specialized nucleosomal structures on methylated DNA helps to explain the capacity of methylated DNA segments to silence transcription more effectively than conventional chromatin. Specialized nucleosomes also provide a potential molecular mechanism for the stable propagation of DNA methylation-dependent transcriptional silencing through cell division.
Alu sequences are frequently encountered during study of human genomic nucleic acid and form a major component of repetitive DNA. This review describes the origin of Alu sequences and their subsequent amplification and evolution into distinct subfamilies. In recent years a number of different functional roles for Alu sequences have been described. The multiple influences of Alu sequences on RNA polymerase II-mediated gene expression and the presence of Alu sequences in RNA polymerase III-generated transcripts are discussed.
Transcripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5'-UTR sequences hybridized to an RNA of approximately 300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5'-UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The approximately 300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs.
Identification and characterization of the regulatory elements of the human aggrecan gene are necessary first steps in addressing the molecular mechanisms through which the gene is regulated. Using luciferase reporter constructs driven by the human aggrecan promoter or the cytomegalovirus promoter, the 5'- and 3'-untranslated regions of the human aggrecan gene were found to regulate gene expression transcriptionally in a promoter- and/or cell type-specific manner. Independent of cell type, the 5'-untranslated region was inhibitory with respect to the cytomegalovirus promoter, but it was stimulatory to the human aggrecan promoter. The 5'-untranslated region inhibited the cytomegalovirus promoter by approximately 60% in both chondrocytes and NIH 3T3 cells, but it stimulated the activity of the human aggrecan promoter about 8-fold in chondrocytes and 40-fold in NIH 3T3 cells. In contrast, the 3'-untranslated region inhibited the activities of the human aggrecan promoter by 40-70% in both cell types, but it stimulated the cytomegalovirus promoter activities by 50-60% in NIH 3T3 cells and inhibited its activity by 70% in chondrocytes. The differential effects of the untranslated regions on the two types of promoters may be a reflection of differences in regulation of TATA-less promoters, such as the human aggrecan promoter, and TATA-containing promoters, such as the cytomegalovirus promoter.
Available data on possible genetic impacts of mammalian retroposons are reviewed. Most important is the growing number of established examples showing the involvement of retroposons in modulation of expression of protein-coding genes transcribed by RNA polymerase II (Pol II). Retroposons contain conserved blocks of nucleotide sequence for binding of some important Pol II transcription factors as well as sequences involved in regulation of stability of mRNA. Moreover, these mobile genes provide short regions of sequence homology for illegitimate recombinations, leading to diverse genome rearrangements during evolution. Therefore, mammalian retroposons representing a significant fraction of noncoding DNA cannot be considered at present as junk DNA but as important genetic symbionts driving the evolution of regulatory networks controlling gene expression.
The methylation of CpG islands is often equated with transcriptional inactivity and there is overwhelming evidence that this is the case for islands located in gene promoters. Such methylation is probably part of a mechanism to permanently silence the activities of genes, including those on the inactive X chromosome. Not all CpG islands and methylation sites are located in known promoters; several tissue-specific and imprinted genes have CpG islands located at considerable distances downstream of transcription initiation sites, and many genes have multiple promoters. Methylation of CpG islands downstream of transcription initiation does not block elongation in mammalian cells. This has given rise to an interesting paradox in which methylation in the transcribed region is often correlated with expression, in contrast to the inverse correlation seen at the site of transcriptional initiation. The methylation paradox might be resolved if it is hypothesized that transcription through a CpG island facilitates de novo methylation.
'BLAST 2 Sequences', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 Sequences' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.++ +html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).
Nuclear respiratory factor 1 (NRF-1) is a nuclear transcription factor that has been implicated in the nuclear control of respiratory chain expression in mammalian cells. Here, we demonstrate that a complex pattern of alternative splicing contributes to sequence heterogeneity within the human NRF-1 5'-untranslated region (UTR). At least six different 5'-UTR exons (UTRs 1-6) were detected in NRF-1 transcripts. These exons were mapped to human NRF-1 genomic clones and their sequences, including donor and acceptor splice junctions, determined. Two of the human UTR exons were derived from insertions of Alu-sq family members into the NRF-1 locus. The distance between the transcription initiation sites in UTR1 and the first protein coding exon is approx. 47kb, bringing the total length of the human NRF-1 gene to approx. 104kb. In contrast to human, only two UTR exons were found in mouse. The mouse UTR1 sequence obtained is identical to human UTR1, but mouse UTR2 bears no resemblance to any of the human exons. Mutations within human UTR1 modulate NRF-1 expression by interfering with mRNA translational efficiency in transfected cells and in an in vitro translation system. The effects of the mutations are proportional to their ability to disrupt predicted mRNA secondary structures within UTR1. Thus, the unusually high sequence conservation within UTR1 in part reflects selective constraints on translational expression.
The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle involved in the co-translational targeting of proteins to membranes. Crystal structures are now available for three protein-RNA subcomplexes from the SRP, which give insights into fundamental aspects of protein-RNA recognition, the assembly of stable ribonucleoprotein particles and the mechanism of action of the SRP.
The impact of L1 retrotransposons on the human genome
- Kazazian
Kazazian, H. H. J., and Moran, J. V. (1998). The impact of L1 retrotransposons on the
human genome. Nat. Genet. 19: 19-24.
Human a2(VI) collagen gene
- Saitta
Saitta, B., Timpl, R., and Chu, M.-L. (1992). Human a2(VI) collagen gene. J. Biol. Chem.
267: 6188–6196.
Structure and assembly of the Alu domain of the mammalian signal recognition particle
- Weichenrieder
How does DNA methylation repress transcription
- Kass