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Alternative splicing of human U2AF1 gene. A, structure of human U2AF1 gene; exons are represented by boxes and introns by lines. A homologue of chicken exon Ab is present in the sequence of human U2AF1 second intron. The alternative splicing patterns are indicated (a– c); B, representation of the three distinct alternative splicing products of human U2AF1 gene; form a corresponds to the cDNA reported by Zhang et al. (19); in form b exon Ab substitutes exon 3; and in form c exon Ab is included between exons 2 and 3, introducing of a premature stop codon (TAA). Sequences of exons 3 and Ab are shown, and the different nucleotides between both are highlighted in gray. C, comparison of the amino acid sequences translated from the three alternative splicing products. The peptide sequences coded by exons 3 and Ab are boxed and differ in 7 amino acid residues. Nonsimilar amino acids are depicted as dark gray, and similar amino acids are depicted as light gray. The asterisk indicates a stop codon.
Source publication
U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U...
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... Alterna- tive Splicing of Human U2AF1 Gene-In an attempt to iden- tify sequences corresponding to exons Aa and Ab in the hu- man U2AF1 gene, we performed BLAST searches against the human chromosome 21 genomic contig (accession number NT_030188). This allowed us to identify a sequence homo- logue to chicken exon Ab in the second intron of hU2AF1 (Fig. 3A), but we could not find any sequence homologue to chicken exon Aa in either the hU2AF1 gene or ESTs data base. Sequence analysis of human ESTs led us to group mRNAs according to three different alternative splicing patterns (Fig. 3, A and B). Pattern a corresponds to the mRNA previ- ously described (19). In splicing pattern b, exon Ab ...
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... NT_030188). This allowed us to identify a sequence homo- logue to chicken exon Ab in the second intron of hU2AF1 (Fig. 3A), but we could not find any sequence homologue to chicken exon Aa in either the hU2AF1 gene or ESTs data base. Sequence analysis of human ESTs led us to group mRNAs according to three different alternative splicing patterns (Fig. 3, A and B). Pattern a corresponds to the mRNA previ- ously described (19). In splicing pattern b, exon Ab substi- tutes exon 3 with no alteration of the reading frame. Trans- lation of this type of transcript gives rise to a protein product very similar to the known U2AF 35 , with only 7 different amino acid residues (Fig. 3C). Finally, as result ...
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... splicing patterns (Fig. 3, A and B). Pattern a corresponds to the mRNA previ- ously described (19). In splicing pattern b, exon Ab substi- tutes exon 3 with no alteration of the reading frame. Trans- lation of this type of transcript gives rise to a protein product very similar to the known U2AF 35 , with only 7 different amino acid residues (Fig. 3C). Finally, as result of splicing pattern c, exon Ab is included between exons 2 and 3 and induces a frameshift that introduces a premature termina- tion codon (TAA), similarly to what is observed in chicken class III cDNAs (see Fig. 2B). We shall hereafter refer to the isoform produced by each alternative splicing pattern as U2AF 35 a, ...
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... PCR was carried out using primers flanking the alternative regions, and the ampli- fied DNA was then digested with HinfI to allow the discrimi- nation of the three isoforms. As depicted in Fig. 4, 48 h after transfection of HeLa cells with siRNAs against hUpf1, there is a specific increase in the steady-state levels of the U2AF 35 c isoform (Fig. 4, lane 3), compared with control transfected cells (Fig. 4, lane 2). Western blot analysis confirmed that the level of Upf1 protein was significant reduced in the transfected cells (not shown). From this we conclude that U2AF1 pre-mRNAs that undergo splicing pattern c, which includes exon Ab be- tween exons 2 and 3 introducing a premature ...
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... to deplete U2AF from HeLa nuclear extracts, as described previously (38). The extract obtained from the column odTNE was unable to support in vitro splic- ing assays unless complemented with U2AF activity. For some splicing substrates that contain strong 3 splice site signals, such as AdML, complementation can be achieved with U2AF 65 alone (23) (Fig. 5D, compare lanes 2 and 3). However, simulta- neous addition of U2AF 65 and U2AF 35 a significantly enhances the efficiency of AdML pre-mRNA splicing (Fig. 5D, compare lanes 3 and 4). Although U2AF 35 b can also enhance splicing activity, this isoform appears to be less efficient than U2AF 35 a (compare lanes 4 and ...
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... ing assays unless complemented with U2AF activity. For some splicing substrates that contain strong 3 splice site signals, such as AdML, complementation can be achieved with U2AF 65 alone (23) (Fig. 5D, compare lanes 2 and 3). However, simulta- neous addition of U2AF 65 and U2AF 35 a significantly enhances the efficiency of AdML pre-mRNA splicing (Fig. 5D, compare lanes 3 and 4). Although U2AF 35 b can also enhance splicing activity, this isoform appears to be less efficient than U2AF 35 a (compare lanes 4 and ...
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... the chicken, human, and mouse U2AF1 genes, we identi- fied a novel exon, referred to as exon Ab. The simultaneous inclusion of exon Ab and exon 3 by alternative splicing intro- duces an in-frame premature termination codon, and we have classified this type of mRNA as isoform U2AF 35 c (Fig. 3B). Our experimental data further indicate that human U2AF 35 c mRNAs are targeted to NMD, a mechanism by which cells selectively recognize and degrade mRNAs that contain prema- ture termination codons (for recent reviews see Refs. 52-54). Although NMD was classically considered to represent a major surveillance mechanism that destroys ...
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Citations
... During pre-mRNA maturation, the splicing factor SF1 is recruited and binds to a specific adenine located on the intron, recognizing the branch point sequence at 3'splice site (Voith Von Voithenberg et al., 2016). Then, U2AF35 serves as the auxiliary factor responsible for forming a complex with U2AF65 by establishing a stable interaction between U2 snRNA and the 3' region of pre-mRNA intron during the initiation of spliceosome recruitment (Pacheco et al., 2004). ...
Host manipulation is a common strategy for invading pathogens. Trypanosoma cruzi, the causative agent of Chagas Disease, lives intracellularly within host cells. During infection, parasite-associated modifications occur to the host cell metabolism and morphology. However, little is known about the effect of T. cruzi infection on the host cell nucleus and nuclear functionality. Here, we show that T. cruzi can modulate host transcription and splicing machinery in non-professional phagocytic cells during infection. We found that T. cruzi regulates host RNA polymerase II (RNAPII) in a time-dependent manner, resulting in a drastic decrease in RNAPII activity. Furthermore, host cell ribonucleoproteins associated with mRNA transcription (hnRNPA1 and AB2) are downregulated concurrently. We reasoned that T. cruzi may hijack the host U2AF35 auxiliary factor, a key regulator for RNA processing, as a strategy to affect the splicing machinery activities directly. In support of our hypothesis, we carried out in vivo splicing assays using an adenovirus E1A pre-mRNA splicing reporter, showing that intracellular T. cruzi directly modulates the host cells by appropriating U2AF35. For the first time, our results provide evidence of a complex and intimate molecular relationship between T. cruzi and the host cell nucleus during infection.
... To determine the effect of U2af1 on hematopoietic development, we genetically manipulated the second Zn that affects all the described U2af1 isoforms [30,31] to create a mutant truncated allele at the C-terminus by a CRISPR−Cas9 system (Figure 1a). We first analyzed the 5-nucleotide deletion in the DNA of control WT and heterozygous U2af1 mut/+ mice by Sanger sequencing (Figure 1a). ...
Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3′ splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+ Tet2−/− mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+ Tet2−/− mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.
... In addition, there are distinct mRNA isoforms of U2AF1, 1 of which includes a premature termination codon that targets the resulting U2AF1 mRNA for nonsensemediated mRNA decay. 31 However, U2AF1 S34F/Q157 doublemutant clones did not differ from U2AF1 WT clones in their usage of U2AF1 isoforms (supplemental Figure 5B). Consistent with these findings, patients with U2AF1 S34F/Q157 double mutations harbored differential cassette exon splicing events, characteristic of both the U2AF1 S34F and U2AF1 Q157 single-mutation states ( Figure 3J). ...
... The discovery that cells containing hotspot mutations in RNA splicing factors are intolerant of additional genetic perturbations in splicing led to a therapeutic effort to modulate splicing as a novel form of therapy for splicing factor mutant leukemias. 31 However, the allele-specific effects of splicing factor mutations on vulnerability to additional genetic perturbations to splicing identified in our study suggest the possibility that response to such therapies may vary based on the exact mutant allele present. For example, cells bearing SF3B1 K700E and SRSF2 P95H/L/R substitutions may be more sensitive to splicing modulatory drugs than U2AF1 S34 or U2AF1 Q157 mutations or rarer mutant alleles in SRSF2 or SF3B1, which have less prominent effects on RNA splicing than K700E or P95H/L/R substitutions. ...
Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are amongst the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. Here we performed bulk and single cell analyses of myeloid malignancy patients harboring >2 splicing factor mutations to understand the frequency and basis for the co-existence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4,231 patients (q<0.001), 0.85% harbored two concomitant bona fide splicing factor mutations, ~50% of which were present in the same individual cells. However, the distribution of mutations in double mutants deviated from those in single mutants with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in double mutants had reduced effects on RNA splicing and/or binding compared to the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating molecular effects of splicing factor mutations as well as their co-occurrences/exclusivities with one another.
... These two duplicated tandem exons (3a and 3b (formerly designated as exon Ab)) are mutually exclusive in splicing and yield two highly similar isoforms, U2AF1a and U2AF1b. They are evolutionary conserved and only differ by seven amino acids in the final protein products (97.1% identity) (7,8). It has been shown that U2AF1a is more abundant than U2AF1b in various cell lines and tissues (7)(8)(9). ...
... They are evolutionary conserved and only differ by seven amino acids in the final protein products (97.1% identity) (7,8). It has been shown that U2AF1a is more abundant than U2AF1b in various cell lines and tissues (7)(8)(9). Because of inherent similarities and biased expression of U2AF1a, studies on the functional differences between U2AF1 isoforms are largely lacking. ...
... It is known that three transcripts are transcribed from the U2AF1 gene. Evolutionarily conserved, mutually exclusive tandem exons drive the transcription of the two isoform transcripts, U2AF1a and U2AF1b, and the inclusion of both exons produce U2AF1c transcript which is subjected to nonsense-mediated mRNA decay (8). The differences between two U2AF1 isoforms encoded by alternative exon 3 usage occur at the atypical RNA recognition motif which is involved in the dimerization with U2AF2 (14). ...
U2 auxiliary factor 1 (U2AF1) functions in 3'-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms' inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3'-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5'-untranslated region (5'-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5'-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control.
... Additionally, the U2AF1c isoform is targeted by RNA surveillance for the introduction of a premature termination codon. 37 In addition, studies have demonstrated that U2AF1a is essential for HeLa cell division and that knockdown of U2AF1a, alone or in conjunction with U2AF1b, has a more significant effect than knockdown of U2AF1b alone. 24 increases p53-dependent apoptosis by impairing hnRNPC regulation of p53 activity. ...
U2AF1 (U2AF35) is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP (small nuclear ribonucleoproteins) of the spliceosome. Here, we examined the function of U2AF1 in human erythropoiesis. First, we examined the expression of U2AF1 during in vitro human erythropoiesis and showed that U2AF1 was highly expressed in the erythroid progenitor burst‐forming‐unit erythroid (BFU‐E) cell stage. A colony assay revealed that U2AF1 knockdown cells failed to form BFU‐E and colony‐forming‐unit erythroid (CFU‐E) colonies. Our results further showed that knockdown of U2AF1 significantly inhibited cell growth and induced apoptosis in erythropoiesis. Additionally, knockdown of U2AF1 also delayed terminal erythroid differentiation. To explore the molecular basis of the impaired function of erythroid development, RNA‐seq was performed and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage, were involved, with the p53 signalling pathway showing the greatest involvement. Western blot analysis revealed an increase in the protein levels of downstream targets of p53 following U2AF1 knockdown. The data further showed that depletion of U2AF1 altered alternatively spliced apoptosis‐associated gene transcripts in CFU‐E cells. Our findings elucidate the role of U2AF1 in human erythropoiesis and reveal the underlying mechanisms.
... It has been increasingly reported over past years that NMD is not only dedicated to the destruction of PTC-containing mRNAs that appear as a result of nonsense mutations or splicing errors, but that it also plays a key role in regulating the expression of a broad class of physiological transcripts (8,9). Targets of NMD include tissuespecific transcripts (10), transcripts with mutually exclusive exons (11), mRNAs with upstream open reading frames (uORFs) and long 3 -untranslated regions (UTRs) (12), and transcripts emanating from transposons and retroviruses (13). The mechanism, in which the cell employs alternative splicing (AS) coupled with NMD to downregulate the abundance of mRNA transcripts, shortly termed AS-NMD (14) (also referred to as regulated unproductive splicing and translation (8) or unproductive splicing (15)), is found in all eukaryotes that have been studied to date and often exhibits a high degree of evolutionary conservation (16,17). ...
... Since 67 is not a multiple of three, simultaneous inclusion of exons 3a and 3b, or simultaneous skipping of both, leads to a frameshift. Studies have reported that simultaneous inclusion of these two exons indeed leads to the degradation of the corresponding transcript isoform by NMD (11). We find that exon 3b, which is located downstream of exon 3a, reacts positively to UPF1/XRN1 co-depletion ( = 0.18 with P < 10 −3 ), and negatively to U2AF1 depletion ( = −0.244 ...
Nonsense-mediated decay (NMD) is a eukary-otic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic analysis integrating three data sources, eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such autoregulatory feedback loops. We show that RBPs frequently bind their own pre-mRNAs, their exons respond prominently to NMD pathway disruption , and that the responding exons are enriched with nearby eCLIP peaks. We confirm previously proposed models of autoregulation in SRSF7 and U2AF1 genes and present two novel models, in which (i) SFPQ binds its mRNA and promotes switching to an alternative distal 3-UTR that is targeted by NMD, and (ii) RPS3 binding activates a poison 5-splice site in its pre-mRNA that leads to a frame shift and degradation by NMD. We also suggest specific splicing events that could be implicated in autoregula-tory feedback loops in RBM39, HNRNPM, and U2AF2 genes. The results are available through a UCSC Genome Browser track hub.
... It has been increasingly reported over past years that NMD is not only dedicated to the destruction of PTC-containing mRNAs that appear as a result of nonsense mutations or splicing errors, but that it also plays a key role in regulating the expression of a broad class of physiological transcripts (8,9). Targets of NMD include tissuespecific transcripts (10), transcripts with mutually exclusive exons (11), mRNAs with upstream open reading frames (uORFs) and long 3 -untranslated regions (UTRs) (12), and transcripts emanating from transposons and retroviruses (13). The mechanism, in which the cell employs alternative splicing (AS) coupled with NMD to downregulate the abundance of mRNA transcripts, shortly termed AS-NMD (14) (also referred to as regulated unproductive splicing and translation (8) or unproductive splicing (15)), is found in all eukaryotes that have been studied to date and often exhibits a high degree of evolutionary conservation (16,17). ...
... Since 67 is not a multiple of three, simultaneous inclusion of exons 3a and 3b, or simultaneous skipping of both, leads to a frameshift. Studies have reported that simultaneous inclusion of these two exons indeed leads to the degradation of the corresponding transcript isoform by NMD (11). We find that exon 3b, which is located downstream of exon 3a, reacts positively to UPF1/XRN1 co-depletion ( = 0.18 with P < 10 −3 ), and negatively to U2AF1 depletion ( = −0.244 ...
Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic framework to identify novel such autoregulatory feedback loops by combining eCLIP assays for a large panel of RBPs with the data on shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq. We show that RBPs frequently bind their own pre-mRNAs and respond prominently to NMD pathway disruption. Poison and essential exons, i.e., exons that trigger NMD when included in the mRNA or skipped, respectively, respond oppositely to the inactivation of NMD pathway and to the depletion of their host genes, which allows identification of novel autoregulatory mechanisms for a number of human RBPs. For example, SRSF7 binds its own pre-mRNA and facilitates the inclusion of two poison exons; SFPQ binding promotes switching to an alternative distal 3'-UTR that is targeted by NMD; RPS3 activates a poison 5'-splice site in its pre-mRNA that leads to a frame shift; U2AF1 binding activates one of its two mutually exclusive exons, leading to NMD; TBRG4 is regulated by cluster splicing of its two essential exons. Our results indicate that autoregulatory negative feedback loop of alternative splicing and NMD is a generic form of post-transcriptional control of gene expression.
... U2AF65 has been shown to compete with PTBP1 for binding to polypyrimidine tracts at alternatively spliced exons and to promote splicing in the absence of PTBP1. (Pacheco et al., 2004;Sauli ere et al., 2006;Shao et al., 2014;Sharma, Falick, & Black, 2005). Other interesting candidates to explore include RBM4, RBM24, and MBNL1, which all have determinant roles in muscle-specific splicing (Konieczny, Stepniak-Konieczna, & Sobczak, 2014;Lin & Tarn, 2011;Yang et al., 2014). ...
Background
Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intron mutation in the iron‐sulfur cluster assembly (ISCU) gene. The mutation results in aberrant splicing, where part of the intron is retained in the final mRNA transcript, giving rise to a truncated nonfunctional ISCU protein. Using an ISCU mini‐gene system, we have previously shown that PTBP1 can act as a repressor of the mis‐splicing of ISCU, where overexpression of PTBP1 resulted in a decrease of the incorrect splicing. In this study, we wanted to, in more detail, analyze the role of PTBP1 in the regulation of endogenous ISCU mis‐splicing.
Methods
Overexpression and knockdown of PTBP1 was performed in myoblasts from two HML patients and a healthy control. Quantification of ISCU mis‐splicing was done by qRTPCR. Biotinylated ISCU RNA, representing wildtype and mutant intron sequence, was used in a pull‐down assay with nuclear extracts from myoblasts. Levels of PTBP1 in human cell lines and mice tissues were analyzed by qRTPCR and western blot.
Results
PTBP1 overexpression in HML patient myoblasts resulted in a substantial decrease of ISCU mis‐splicing while knockdown of PTBP1 resulted in a drastic increase. The effect could be observed in both patient and control myoblasts. We could also show that PTBP1 interacts with both the mutant and wild‐type ISCU intron sequence, but with a higher affinity to the mutant sequence. Furthermore, low levels of PTBP1 among examined mouse tissues correlated with high levels of incorrect splicing of ISCU.
Conclusion
Our results show that PTBP1 acts as a dominant repressor of ISCU mis‐splicing. We also show an inverse correlation between the levels of PTBP1 and ISCU mis‐splicing, suggesting that the high level of mis‐splicing in the skeletal muscle is primarily due to the low levels of PTBP1.
... Intriguingly, some cases have shown that duplicated genes in one species are very similar to alternative spliced isoforms in another species (Pacheco, et al. 2004). Meanwhile, recent studies have shown large gene families are lacked of splicing isoforms and vice versa (Su, et al. 2006). ...
The evolution of duplicated genes after polyploidization has been the subject of many evolutionary biology studies. Potato (Solanum tuberosum) and tomato (Solanum lycopersicum) are the first two sequenced genomes of asterids, and share a common polyploidization event. However, the epigenetic role of DNA methylation on the evolution of duplicated genes derived from polyploidization is not fully understood. Here, we explore the role of the DNA methylation in the evolution of duplicated genes in potato and tomato. The overall levels of DNA methylation are different, although patterns of DNA methylation are similar in potato and tomato. Different types of duplicated genes can display different methylation patterns in potato and tomato. In addition, we found that differences in the methylation levels between duplicated genes were associated with gene expression divergence. In particular, for the majority of duplicated gene pairs, one copy is always hyper- or hypo-methylated compared to the other copy across different tomato fruit ripening stages, and these genes are enriched for specific function related to transcription factor activity. Furthermore, transcription of hundreds of duplicated transcription factors was shown to be regulated by DNA methylation during fruit ripening stages in tomato, some of which are well-known fruit ripening transcription factors. Taken together, our results support the notion that DNA methylation may facilitate divergent evolution of duplicated genes and play roles in important biological processes such as tomato fruit ripening.
... An example of conserved AS in the 3'UTR region was identified for U2AF35, that is a component of the U2AF (U2 snRNP auxiliary factor) heterodimer, an essential pre-mRNA splicing factor. U2AF35 plays critical roles in the recognition of the 3′-splicing [54]. In addition human U2AF35 is implicated in the determination of mRNAs 3'UTR-length; mutated U2AF35 results in longer 3'UTR of certain genes [55]. ...
Background
The vast diversification of proteins in eukaryotic cells has been related with multiple transcript isoforms from a single gene that result in alternative splicing (AS) of primary transcripts. Analysis of RNA sequencing data from expressed sequence tags and next generation RNA sequencing has been crucial for AS identification and genome-wide AS studies. For the identification of AS events from the related legume species Phaseolus vulgaris and Glycine max, 157 and 88 publicly available RNA-seq libraries, respectively, were analyzed. ResultsWe identified 85,570 AS events from P. vulgaris in 72% of expressed genes and 134,316 AS events in 70% of expressed genes from G. max. These were categorized in seven AS event types with intron retention being the most abundant followed by alternative acceptor and alternative donor, representing ~75% of all AS events in both plants. Conservation of AS events in homologous genes between the two species was analyzed where an overrepresentation of AS affecting 5’UTR regions was observed for certain types of AS events. The conservation of AS events was experimentally validated for 8 selected genes, through RT-PCR analysis. The different types of AS events also varied by relative position in the genes. The results were consistent in both species. Conclusions
The identification and analysis of AS events are first steps to understand their biological relevance. The results presented here from two related legume species reveal high conservation, over ~15–20 MY of divergence, and may point to the biological relevance of AS.
