Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Special Characteristics of Alternative 3′ and 5′ Splicing Sites Human–mouse conserved 3′ and 5′ alternative splicing events (A3Es and A5Es, respectively) were divided into two subgroups according to their relative usage, in which the alternative splice site that is supported by most EST/cDNA is called Major, whereas the less-selected site is the Minor (see Materials and Methods). Splice site score of the 3′ and 5′ splice sites of constitutive, cassette (exon skipping), alternative 3′, and alternative 5′ exons was calculated using the “Analyzer Splice Tool” server (http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.htm). Human exon scores are shown above the exon/intron junction scheme (see Figure S1 for mouse scores). Major/Minor splice site is indicated below each splice site. Exon sequence is represented by a yellow box and the alternative sequence (extension) by a light blue box. Introns are represented by black lines; canonical splice sites are shown in bold.
Source publication
Alternative 3' and 5' splice site (ss) events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the c...
Similar publications
The +1169A allele of the A/T single nucleotide polymorphism (SNP; rs2665802), located within intron 4 of the human growth hormone 1 ( GH1 ) gene, has been associated with reduced levels of circulating GH and insulin-like growth factor 1, a reduced risk of colorectal cancer and a predisposition to osteoporosis. Whether this intronic SNP is itself th...
Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome caused by the NF2 tumor suppressor gene. However, the NF2 mutation characteristics in Korean patients are not sufficiently understood. In this study, we conducted a comprehensive mutational analysis in 7 Korean NF2 patients by performing direct sequencing and gene-dosage assessment.
W...
Citations
... Alternative 5' splice site and alternative 3`splice site are less common compared to exon skipping, and both AS mechanisms represent an intermediate state between constitutive and alternative cassette exons. The respective exon has on one site a fixed splice site and on the other site two or more competing splice sites, leading after splicing to different lengths of these regions (15). The fifth mechanism of AS forming "mutually exclusive exons" describes a splicing event with coordinated exon splicingthus, one exon or one group of exons is retained, while the other exon or group of exons is spliced out. ...
Alternative splicing (AS) is an important molecular biological mechanism regulated by complex mechanisms involving a plethora of cis and trans-acting elements. Furthermore, AS is tissue specific and altered in various pathologies, including infectious, inflammatory, and neoplastic diseases. Recently developed immuno-oncological therapies include monoclonal antibodies (mAbs) and chimeric antigen receptor (CAR) T cells targeting, among others, immune checkpoint (ICP) molecules. Despite therapeutic successes have been demonstrated, only a limited number of patients showed long-term benefit from these therapies with tumor entity-related differential response rates were observed. Interestingly, splice variants of common immunotherapeutic targets generated by AS are able to completely escape and/or reduce the efficacy of mAb- and/or CAR-based tumor immunotherapies. Therefore, the analyses of splicing patterns of targeted molecules in tumor specimens prior to therapy might help correct stratification, thereby increasing therapy success by antibody panel selection and antibody dosages. In addition, the expression of certain splicing factors has been linked with the patients’ outcome, thereby highlighting their putative prognostic potential. Outstanding questions are addressed to translate the findings into clinical application. This review article provides an overview of the role of AS in (tumor) diseases, its molecular mechanisms, clinical relevance, and therapy response.
... Due to the 413 relatively high Kirrel3 exon-count, it is not surprising that Kirrel3 isoform diversity arises from many 414 different types of splicing events including, exon skipping, alternative 3' exon splicing (mouse and 415 human), and alternative 5' exon splicing (human). These three forms of alternative splicing occur at 416 frequencies for Kirrel3 that roughly match the proportions typical for mammalian splice events (38% 417 exon skipping, 18% alternative 3' splicing, 8% alternative 5' splicing) (Koren, Lev-Maor et al. 2007). 418 419 Importantly, we provide evidence that even rare Kirrel3 isoforms present in our full-length transcript 420 data set make detectable levels of mRNA and protein in the mouse brain. ...
Kirrel3 is a cell-adhesion molecule that instructs the formation of specific synapses during brain development in mouse and Kirrel3 variants may be risk factors for autism and intellectual disabilities in humans. Kirrel3 is predicted to undergo alternative splicing but brain isoforms have not been studied. Here, we present the first in-depth characterization of Kirrel3 isoform diversity in brain using targeted, long-read mRNA sequencing of mouse hippocampus. We identified 19 isoforms with predicted transmembrane and secreted forms and show that even rare isoforms generate detectable protein in the brain. We also analyzed publicly-available long-read mRNA databases from human brain tissue and found 11 Kirrel3 isoforms that, similar to mouse, encode transmembrane and secreted forms. In mice and humans, Kirrel3 diversity arises from alternative, independent use of protein-domain coding exons and alternative early translation-stop signals. Intriguingly, the alternatively spliced exons appear at branch points in the chordate phylogenetic tree, including one exon only found in humans and their closest living relatives, the great apes. Together, these results validate a simple pipeline for analyzing isoform diversity in genes with low expression and suggest that Kirrel3 function is fine-tuned by alternative splicing and may play a role in brain evolution.
Significance Statement
Kirrel3 is an important molecule for synapse and circuit formation with gene variants that are associated with neurodevelopmental disorders, yet Kirrel3 function remains largely unknown. Here, we report new isoforms of mouse and human Kirrel3, including secreted and transmembrane forms, that suggest a diverse repertoire of Kirrel3 actions. Importantly, we identified a new Kirrel3 exon only present in humans and the other great apes with potential to play an important role in circuit formation unique to these species.
... Gene transcription produces a pre-mRNA molecule that can be subjected to a variety of splicing events and generate a plethora of spliced variants due to the presence in many genes of skipped exons (SEs), alternative 3 0 splice sites (A3SSs), alternative 5'splice sites (A5SSs), mutually exclusive exons (MXEs), and intron retention (IR). 1,2 Thus, AS of pre-mRNA molecules can dramatically expand the complexity of the transcriptome and hence expand the diversity of the proteome. 3,4 In humans, 90%-95% of multi-exon genes produce alternatively spliced RNA transcripts, so the widespread use underscores the importance of AS. ...
Alternative splicing (AS) of RNA molecules is a key contributor to transcriptome diversity. In humans, 90%-95% of multi-exon genes produce alternatively spliced RNA transcripts. Therefore, every single gene has the opportunity of producing multiple splice variants, including long non-coding RNA (lncRNA) genes that undergo RNA maturation steps such as conventional and alternative splicing. Emerging evidence suggests significant roles for these lncRNA splice variants in many aspects of cell biology. Differential changes in expression of specific lncRNA splice variants have also been associated with many diseases including cancer. This review covers the current knowledge on this emerging topic of investigation. We provide exclusive insights on the AS landscape of lncRNAs and also describe at the molecular level the functional relevance of lncRNA splice variants, i.e., RNA-based differential functions, production of micropeptides, and generation of circular RNAs. Finally, we discuss exciting perspectives for this emerging field and outline the work required to further develop research endeavors in this field.
... As detailed previously, CRISPy TAKO mutants harbor variable mutations (83) and at some loci such as Pitt3, this can lead to expression of novel transcripts from the targeted locus. This could be the result of the mutations impacting the 5' splice site(s), or mutating splicer enhancer/repressor binding sites and therefore shifting splicing dynamics (97)(98)(99)(100)(101). Analysis of downstream sequences in Pitt4 mutants revealed that the downstream cDNA amplicon was readily detected in control and all TAKOs analyzed ( Figure 6F; middle panel). ...
The molecular mechanisms regulating the development and progression of alcohol use disorder (AUD) are largely unknown. While noncoding RNAs have previously been implicated as playing key roles in AUD, long-noncoding RNA (lncRNA) remains understudied in relation to AUD. In this study, we first identified ethanol-responsive lncRNAs in the mouse hippocampus that are transcriptional network hub genes. Microarray analysis of lncRNA, miRNA, circular RNA, and protein coding gene expression in the hippocampus from chronic intermittent ethanol vapor- or air- (control) exposed mice was used to identify ethanol-responsive competing endogenous RNA (ceRNA) networks. Highly interconnected lncRNAs (genes that had the strongest overall correlation to all other dysregulated genes identified) were ranked. The top four lncRNAs were novel, previously uncharacterized genes named Gm42575, 4930413E15Rik, Gm15767, and Gm33447, hereafter referred to as Pitt1, Pitt2, Pitt3, and Pitt4, respectively. We subsequently tested the hypothesis that CRISPR/Cas9 mutagenesis of the putative promoter and first exon of these lncRNAs in C57BL/6J mice would alter ethanol drinking behavior. The Drinking in the Dark (DID) assay was used to examine binge-like drinking behavior, and the Every-Other-Day Two-Bottle Choice (EOD-2BC) assay was used to examine intermittent ethanol consumption and preference. No significant differences between control and mutant mice were observed in the DID assay. Female-specific reductions in ethanol consumption were observed in the EOD-2BC assay for Pitt1, Pitt3, and Pitt4 mutant mice compared to controls. Male-specific alterations in ethanol preference were observed for Pitt1 and Pitt2. Female-specific increases in ethanol preference were observed for Pitt3 and Pitt4. Total fluid consumption was reduced in Pitt1 and Pitt2 mutants at 15% v/v ethanol and in Pitt3 and Pitt4 at 20% v/v ethanol in females only. We conclude that all lncRNAs targeted altered ethanol drinking behavior, and that lncRNAs Pitt1, Pitt3, and Pitt4 influenced ethanol consumption in a sex-specific manner. Further research is necessary to elucidate the biological mechanisms for these effects. These findings add to the literature implicating noncoding RNAs in AUD and suggest lncRNAs also play an important regulatory role in the disease.
... Exonization of a transposable element that lies within an intron provides new cassette exons, but this does not explain other types of alternative splicing. Notably, the emergence of alternative 5' or 3' splice sites constitutes a mechanism to exonize an intronic region immediately adjacent to an existing exon (Koren et al. 2007). Another potential mechanism would be the emergence of a cleavage and polyadenylation site in the intron downstream of an internal exon. ...
Exonization is the evolutionary process of recruitment of new exonic regions from previously intronic regions. It is a major contributor to the increased complexity of alternative splicing. Here, we explore exonization mediated by the emergence of a novel cleavage-polyadenylation site in an intron. In Xenopus laevis , the tpm1 gene, which encodes muscular tropomyosin, contains alternative terminal exons. In adult muscles and embryonic hearts, exon 9A is joined to the terminal exon 9B. In embryonic somites, it is joined to the exonic region 9’, which is transcribed from the intron immediately downstream of exon 9A. Consequently, exon 9A is either an internal exon when ligated to exon 9B, or a part of a terminal exon along with region 9’. We show here that region 9’ is present only in amphibians and coelacanths. This suggests that it emerged in sarcopterygians and was lost in amniotes. We used antisense morpholino oligonucleotides to mask the regions of tpm1 pre-mRNA that potentially regulate the inclusion of exon 9A9’. This revealed that the definition of exon 9A9’ relies on a weak cleavage-polyadenylation site and an intronic enhancer, but is independent of the 3’ splice site. We demonstrate that RNAs containing exon 9B are toxic in somites. This may have contributed to the evolutionary pressure that led to the exonization of region 9’ in sarcopterygians. These findings reveal the emergence of a novel cleavage-polyadenylation site that avoids the accumulation of a toxic RNA as a novel mechanism for exonization-mediated diversification of terminal exons.
... Recent studies have shown that AS events follow conserved patterns of expression shared across species [24,25], indicating an underlying conserved mechanisms and regulatory sequences related to the genes' splicing programs. Additional observations show that some splicing events encounter divergence in their inclusion rates [26] or divergence in their tissue specific expression rates [27], which alternatively suggests regulatory sequences divergence. ...
Background
In eukaryote transcriptomes, a significant amount of transcript diversity comes from genes’ capacity to generate different transcripts through alternative splicing. Identifying orthologous alternative transcripts across multiple species is of particular interest for genome annotators. However, there is no formal definition of transcript orthology based on the splicing structure conservation. Likewise there is no public dataset benchmark providing groups of orthologous transcripts sharing a conserved splicing structure.
Results
We introduced a formal definition of splicing structure orthology and we predicted transcript orthologs in human, mouse and dog. Applying a selective strategy, we analyzed 2,167 genes and their 18,109 known transcripts and identified a set of 253 gene orthologs that shared a conserved splicing structure in all three species. We predicted 6,861 transcript CDSs (coding sequence), mainly for dog, an emergent model species. Each predicted transcript was an ortholog of a known transcript: both share the same CDS splicing structure. Evidence for the existence of the predicted CDSs was found in external data.
Conclusions
We generated a dataset of 253 gene triplets, structurally conserved and sharing all their CDSs in human, mouse and dog, which correspond to 879 triplets of spliced CDS orthologs. We have released the dataset both as an SQL database and as tabulated files. The data consists of the 879 CDS orthology groups with their detailed splicing structures, and the predicted CDSs, associated with their experimental evidence. The 6,861 predicted CDSs are provided in GTF files. Our data may contribute to compare highly conserved genes across three species, for comparative transcriptomics at the isoform level, or for benchmarking splice aligners and methods focusing on the identification of splicing orthologs. The data is available at https://data-access.cesgo.org/index.php/s/V97GXxOS66NqTkZ.
... Alternative 3 and 5 splicing events constitute at least one-quarter of the known AS events [12]. Alternative 5 splicing sites exons (A5Es) and alternative 3 'splicing sites exons (A3Es) are flanked on one end by a constitutive splice site and on the other end by two (or more) alternative splice sites, resulting in different primary transcripts of the same gene [22,24]. ...
... The most common type of AS is exon skipping, also known as cassette exons [12]. Cassette exons are delimited by splice sites located at the boundaries between mRNAcoding and non-coding sequences and may be included or not in the mature mRNA transcript [21][22][23]. When the exon is retained, the splicing pattern is similar to that of a constitutive gene; however, when it is removed, it is presumably spliced out together with its flanking introns [23,24]. ...
Tau protein is a microtubule-associated protein encoded by the MAPT gene that carries out a myriad of physiological functions and has been linked to certain pathologies collectively termed tauopathies, including Alzheimer’s disease, frontotemporal dementia, Huntington’s disease, progressive supranuclear palsy, etc. Alternative splicing is a physiological process by which cells generate several transcripts from one single gene and may in turn give rise to different proteins from the same gene. MAPT transcripts have been proven to be subjected to alternative splicing, generating six main isoforms in the central nervous system. Research throughout the years has demonstrated that the splicing landscape of the MAPT gene is far more complex than that, including at least exon skipping events, the use of 3′ and 5′ alternative splice sites and, as has been recently discovered, also intron retention. In addition, MAPT alternative splicing has been showed to be regulated spatially and developmentally, further evidencing the complexity of the gene’s splicing regulation. It is unclear what would drive the need for the existence of so many isoforms encoded by the same gene, but a wide range of functions have been ascribed to these Tau isoforms, both in physiology and pathology. In this review we offer a comprehensive up-to-date exploration of the mechanisms leading to the outstanding diversity of isoforms expressed from the MAPT gene and the functions in which such isoforms are involved, including their potential role in the onset and development of tauopathies such as Alzheimer’s disease.
... Dans cette thèse, nous examinons des données issues de l'humain, de la souris et du chien. D'un point de vue évolutif, les lignées de l'humain et de la souris se sont séparées il y a 75 à 80 millions d'années alors que la divergence avec le chien a eu lieu il y a 90 à 110 millions d'années (Koren et al. 2007 ;Scheidt et al. 2017 ;Foley et al. 2016, Figure 1.20). Les données qui concernent ces espèces dans la base de données Ensembl sont présentées dans la Table 1.3. ...
... Après avoir pris en compte l'orthologie décrite des sites fonctionnels, nous pourrions ensuite étudier la divergence ou la conservation de ces séquences impliquées dans la régulation de l'expression des transcrits alternatifs. Des études montrent que certains événements d'épissage rencontrent une divergence dans leurs taux d'inclusion (Xiong et al. 2018) ou une divergence dans leurs taux d'expression spécifiques aux tissus (Koren et al. 2007), ce qui suggère une divergence des séquences régulatrices. Nos données identifient les transcrits alternatifs orthologues et les sites d'épissage orthologues. ...
Les organismes vivants sont capables d’exprimer plusieurs transcrits (ou ARN) alternatifs à partir d’un même gène. Ces transcrits sont responsables des mécanismes de régulation de l’organisme, certains sont traduits en protéine. Détecter l’ensemble des transcrits pouvant être exprimés par un gène est aujourd’hui un problème ouvert auquel de nombreuses méthodes informatiques telles que le séquençage des données de l’ARN, les méthodes d’alignements de séquences épissées ou encore les méthodes de génomiques comparative tentent de répondre. Cette thèse propose une méthode de génomique comparative permettant de comparer la séquence de gènes partagés par plusieurs espèces. Il en résulte une méthode de prédiction de transcrits à une échelle multi-espèces, en s’appuyant sur une structure de graphes. Cette méthode a été appliquée à trois espèces (humain, souris, chien). Elle a permis de prédire un nombre important de transcrits et d’identifier un ensemble de gènes conservés entre les trois espèces et partageant les mêmes structures exoniques et les mêmes CDS.
... In this case, two (or more) splicing events are no longer independent, instead executed or disabled in a coordinated manner, which requires an intensive regulatory mechanism [29]. Alternative 3 or 5 splice sites are often found in close proximity and with high symmetry levels to the constitutive splice site, allowing the conservation of the open reading frame [30]. It is currently estimated that alternative 3 ss and/or 5 ss exons account for about 18% and 8% of alternative transcripts in higher eukaryotes. ...
... In this case, two (or more) splicing events are no longer independent, instead executed or disabled in a coordinated manner, which requires an intensive regulatory mechanism [29]. Alternative 3′ or 5′ splice sites are often found in close proximity and with high symmetry levels to the constitutive splice site, allowing the conservation of the open reading frame [30]. It is currently estimated that alternative 3′ ss and/or 5′ ss exons account for about 18% and 8% of alternative transcripts in higher eukaryotes. ...
Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5′ and 3′ splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.
... There are relatively few wellcharacterized examples of 5'and 3' alternative splicing selection, being most of them related to human diseases (Stoilov et al., 2002;Andre & Cooper, 2016). Taken together 5'and 3' alternative splicing selection have a prevalence of 25% among vertebrate (Wang et al., 2015) and they account for ~ 18% and ~ 8% of the conserved events between human and mouse (Koren, Lev-Maor & Ast, 2007). Here we found that exon 1A' is flanked by a constitutive splice site on one side and by two competing alternative splice sites on the other, resulting in an alternative region (extension) that either is included in the transcript (T4) or excluded (T5). ...
The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5́UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5́RACE-PCR we isolated seven ASIP transcripts with alternative 5’UTR, where exons 1A, 1A’, 1C, 1D, and a novel non-coding exon 1A” were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5’UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3’ splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5’UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level.
Paper is available to download form:
https://authors.elsevier.com/c/1dzg91L~GAlAOq
