Alternative Splicing May Not Be the Key to Proteome Complexity.
Ontology highlight
ABSTRACT:
SUBMITTER: Tress ML
PROVIDER: S-EPMC6526280 | biostudies-literature | 2017 Feb
REPOSITORIES: biostudies-literature
Publications
Alternative Splicing May Not Be the Key to Proteome Complexity.
Tress Michael L ML Abascal Federico F Valencia Alfonso A
Trends in biochemical sciences 20161003 2
Alternative splicing is commonly believed to be a major source of cellular protein diversity. However, although many thousands of alternatively spliced transcripts are routinely detected in RNA-seq studies, reliable large-scale mass spectrometry-based proteomics analyses identify only a small fraction of annotated alternative isoforms. The clearest finding from proteomics experiments is that most human genes have a single main protein isoform, while those alternative isoforms that are identified ...[more]
Similar Datasets
Project description:BackgroundTMEM16A (Anoctamin 1; ANO1) is an eight transmembrane protein that functions as a calcium-activated chloride channel. TMEM16A in human exhibits alternatively spliced exons (6b, 13 and 15), which confer important roles in the regulation of channel function. Mouse Tmem16a is reported to consist of 25 exons that code for a 956 amino acid protein. In this study our aim was to provide details of mouse Tmem16a genomic structure and to investigate if Tmem16a transcript undergoes alternative splicing to generate channel diversity.ResultsWe identified Tmem16a transcript variants consisting of alternative exons 6b, 10, 13, 14, 15 and 18. Our findings indicate that many of these exons are expressed in various combinations and that these splicing events are mostly conserved between mouse and human. In addition, we confirmed the expression of these exon variants in other mouse tissues. Additional splicing events were identified including a novel conserved exon 13b, tandem splice sites of exon 1 and 21 and two intron retention events.ConclusionOur results suggest that Tmem16a gene is significantly more complex than previously described. The complexity is especially evident in the region spanning exons 6 through 16 where a number of the alternative splicing events are thought to affect calcium sensitivity, voltage dependence and the kinetics of activation and deactivation of this calcium-activated chloride channel. The identification of multiple Tmem16a splice variants suggests that alternative splicing is an exquisite mechanism that operates to diversify TMEM16A channel function in both physiological and pathophysiological conditions.
| S-EPMC3170211 | biostudies-literature
Project description:BACKGROUND:Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. MAIN BODY:In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. CONCLUSIONS:An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.
| S-EPMC5840707 | biostudies-literature
Project description:Alternative splicing is a critical determinant of genome complexity and, by implication, is assumed to engender proteomic diversity. This notion has not been experimentally tested in a targeted, quantitative manner. Here, we have developed an integrative approach to ask whether perturbations in mRNA splicing patterns alter the composition of the proteome. We integrate RNA sequencing (RNA-seq) (to comprehensively report intron retention, differential transcript usage, and gene expression) with a data-independent acquisition (DIA) method, SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry), to capture an unbiased, quantitative snapshot of the impact of constitutive and alternative splicing events on the proteome. Whereas intron retention is accompanied by decreased protein abundance, alterations in differential transcript usage and gene expression alter protein abundance proportionate to transcript levels. Our findings illustrate how RNA splicing links isoform expression in the human transcriptome with proteomic diversity and provides a foundation for studying perturbations associated with human diseases.
| S-EPMC5554779 | biostudies-literature
Project description:BackgroundAlternative splicing is the process of selecting different combinations of splice sites to produce variably spliced mRNAs. However, the relationships between alternative splicing prevalence and level (ASP/L) and variations of intron size and organism complexity (OC) remain vague. Here, we developed a robust protocol to analyze the relationships between ASP/L and variations of intron size and OC. Approximately 8 Tb raw RNA-Seq data from 37 eumetazoan species were divided into three sets of species based on variations in intron size and OC.ResultsWe found a strong positive correlation between ASP/L and OC, but no correlation between ASP/L and intron size across species. Surprisingly, ASP/L displayed a positive correlation with mean intron size of genes within individual genomes. Moreover, our results revealed that four ASP/L-related pathways contributed to the differences in ASP/L that were associated with OC. In particular, the spliceosome pathway displayed distinct genomic features, such as the highest gene expression level, conservation level, and fraction of disordered regions. Interestingly, lower or no obvious correlations were observed among these genomic features.ConclusionsThe positive correlation between ASP/L and OC ubiquitously exists in eukaryotes, and this correlation is not affected by the mean intron size of these species. ASP/L-related splicing factors may play an important role in the evolution of OC.
| S-EPMC8614042 | biostudies-literature
Project description:The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB, and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunits, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell-dependent; only one major NF-YC isoform is present in a given cell type. The 37- and 50-kDa isoforms are mutually exclusive, and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-PCRs indicate that P1 has a robust housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage in a p53-dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50-kDa transcript being excluded from P2. Specific functional inactivation of the 37-kDa isoform affects the basal levels of G(1)/S blocking and pro-apoptotic genes but not G(2)/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37-kDa isoform in suppression of the DNA damage-response under growing conditions.
| S-EPMC2797189 | biostudies-literature
Project description:Alternative splicing (AS) plays pivotal roles in regulating plant growth and development, flowering, biological rhythms, signal transduction, and stress responses. However, no studies on AS have been performed in Liriodendron chinense, a deciduous tree species that has high economic and ecological value. In this study, we used multiple tools and algorithms to analyze transcriptome data derived from seven tissues via hybrid sequencing. Although only 17.56% (8,503/48,408) of genes in L. chinense were alternatively spliced, these AS genes occurred in 37,844 AS events. Among these events, intron retention was the most frequent AS event, producing 1,656 PTC-containing and 3,310 non-PTC-containing transcripts. Moreover, 183 long noncoding RNAs (lncRNAs) also underwent AS events. Furthermore, weighted gene coexpression network analysis (WGCNA) revealed that there were great differences in the activities of transcription and post-transcriptional regulation between pistils and leaves, and AS had an impact on many physiological and biochemical processes in L. chinense, such as photosynthesis, sphingolipid metabolism, fatty acid biosynthesis and metabolism. Moreover, our analysis showed that the features of genes may affect AS, as AS genes and non-AS genes had differences in the exon/intron length, transcript length, and number of exons/introns. In addition, the structure of AS genes may impact the frequencies and types of AS because AS genes with more exons or introns tended to exhibit more AS events, and shorter introns tended to be retained, whereas shorter exons tended to be skipped. Furthermore, eight AS genes were verified, and the results were consistent with our analysis. Overall, this study reveals that AS and gene interaction are mutual-on one hand, AS can affect gene expression and translation, while on the other hand, the structural characteristics of the gene can also affect AS. This work is the first to comprehensively report on AS in L. chinense, and it can provide a reference for further research on AS in L. chinense.
| S-EPMC7539066 | biostudies-literature
Project description:Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family.In this study we provide, with in silico and in vitro analyses, the first detailed description of the human multi-transcript RCAN3 locus. Its analysis revealed that it is composed of a multigene system that includes at least 21 RCAN3 alternative spliced isoforms (16 of them identified here for the first time) and a new RCAN3 antisense gene (RCAN3AS). In particular, we cloned RCAN3-1,3,4,5 (lacking exon 2), RCAN3-1a,2,3,4,5, RCAN3-1a,3,4,5, RCAN3-1b,2,3,4,5, RCAN3-1c,2,3,4,5, RCAN3-1c,2,4,5 and RCAN3-1c,3,4,5, isoforms that present a different 5' untranslated region when compared to RCAN3. Moreover, in order to verify the possible 5' incompleteness of previously identified cDNA isoforms with the reference exon 1, ten more alternative isoforms were retrieved. Bioinformatic searches allowed us to identify RCAN3AS, which overlaps in part with exon 1a, on the opposite strand, for which four different RCAN3AS isoforms were cloned.In order to analyze the different expression patterns of RCAN3 alternative first exons and of RCAN3AS mRNA isoforms, RT-PCR was performed in 17 human tissues. Finally, analyses of RCAN3 and RCAN3AS genomic sequences were performed to identify possible promoter regions, to examine donor and acceptor splice sequences and to compare evolutionary conservation, in particular of alternative exon 1 or 1c--exon 2 junctions in different species.The description of its number of transcripts, of their expression patterns and of their regulatory regions can be important to clarify the functions of RCAN3 gene in different pathways and cellular processes.
| S-EPMC3178534 | biostudies-literature
Project description:Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ?150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high-resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (?40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally, ?51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation.
| S-EPMC3371709 | biostudies-literature
Project description:BackgroundThe genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.Methodology/principal findingsWe have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.SignificanceOur observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.
| S-EPMC2868907 | biostudies-literature
Project description:Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.
| S-EPMC5457400 | biostudies-literature