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 tend to be the most biologically plausible: those with the most cross-species conservation and those that do not compromise functional domains. Indeed, most alternative exons do not seem to be under selective pressure, suggesting that a large majority of predicted alternative transcripts may not even be translated into proteins.
Although alternative splicing is well documented at the transcript level, large-scale proteomics experiments identify few alternative isoforms.
Proteomics evidence also suggests that the vast majority of genes have a single dominant splice isoform.
Alternative isoforms detected in proteomics experiments tend to be conserved, are highly enriched in subtle splice events such as mutually exclusively spliced homologous exons and events that do not disrupt functional domains.
Recent large-scale RNA-seq studies have shown that tissue specificity seems to be controlled by gene expression rather than alternative splicing.
Variant calling experiments show that most alternative exons are evolving neutrally, which suggests that most alternative splice events are not evolutionary innovations.
Determining the full complement of protein-coding genes is a key goal of genome annotation. The most powerful approach for confirming protein-coding potential is the detection of cellular protein ...expression through peptide mass spectrometry (MS) experiments. Here, we mapped peptides detected in seven large-scale proteomics studies to almost 60% of the protein-coding genes in the GENCODE annotation of the human genome. We found a strong relationship between detection in proteomics experiments and both gene family age and cross-species conservation. Most of the genes for which we detected peptides were highly conserved. We found peptides for >96% of genes that evolved before bilateria. At the opposite end of the scale, we identified almost no peptides for genes that have appeared since primates, for genes that did not have any protein-like features or for genes with poor cross-species conservation. These results motivated us to describe a set of 2001 potential non-coding genes based on features such as weak conservation, a lack of protein features, or ambiguous annotations from major databases, all of which correlated with low peptide detection across the seven experiments. We identified peptides for just 3% of these genes. We show that many of these genes behave more like non-coding genes than protein-coding genes and suggest that most are unlikely to code for proteins under normal circumstances. We believe that their inclusion in the human protein-coding gene catalogue should be revised as part of the ongoing human genome annotation effort.
Abstract
APPRIS (https://appris.bioinfo.cnio.es) is a well-established database housing annotations for protein isoforms for a range of species. APPRIS selects principal isoforms based on protein ...structure and function features and on cross-species conservation. Most coding genes produce a single main protein isoform and the principal isoforms chosen by the APPRIS database best represent this main cellular isoform. Human genetic data, experimental protein evidence and the distribution of clinical variants all support the relevance of APPRIS principal isoforms. APPRIS annotations and principal isoforms have now been expanded to 10 model organisms. In this paper we highlight the most recent updates to the database. APPRIS annotations have been generated for two new species, cow and chicken, the protein structural information has been augmented with reliable models from the EMBL-EBI AlphaFold database, and we have substantially expanded the confirmatory proteomics evidence available for the human genome. The most significant change in APPRIS has been the implementation of TRIFID functional isoform scores. TRIFID functional scores are assigned to all splice isoforms, and APPRIS uses the TRIFID functional scores and proteomics evidence to determine principal isoforms when core methods cannot.
Transposable elements constitute a large fraction of vertebrate genomes and, during evolution, may be co-opted for new functions. Exonization of transposable elements inserted within or close to host ...genes is one possible way to generate new genes, and alternative splicing of the new exons may represent an intermediate step in this process. The genes TMPO and ZNF451 are present in all vertebrate lineages. Although they are not evolutionarily related, mammalian TMPO and ZNF451 do have something in common-they both code for splice isoforms that contain LAP2alpha domains. We found that these LAP2alpha domains have sequence similarity to repetitive sequences in non-mammalian genomes, which are in turn related to the first ORF from a DIRS1-like retrotransposon. This retrotransposon domestication happened separately and resulted in proteins that combine retrotransposon and host protein domains. The alternative splicing of the retrotransposed sequence allowed the production of both the new and the untouched original isoforms, which may have contributed to the success of the colonization process. The LAP2alpha-specific isoform of TMPO (LAP2α) has been co-opted for important roles in the cell, whereas the ZNF451 LAP2alpha isoform is evolving under strong purifying selection but remains uncharacterized.
Abstract
Most coding genes in the human genome are annotated with multiple alternative transcripts. However, clear evidence for the functional relevance of the protein isoforms produced by these ...alternative transcripts is often hard to find. Alternative isoforms generated from tandem exon duplication-derived substitutions are an exception. These splice events are rare, but have important functional consequences. Here, we have catalogued the 236 tandem exon duplication-derived substitutions annotated in the GENCODE human reference set. We find that more than 90% of the events have a last common ancestor in teleost fish, so are at least 425 million years old, and twenty-one can be traced back to the Bilateria clade. Alternative isoforms generated from tandem exon duplication-derived substitutions also have significantly more clinical impact than other alternative isoforms. Tandem exon duplication-derived substitutions have >25 times as many pathogenic and likely pathogenic mutations as other alternative events. Tandem exon duplication-derived substitutions appear to have vital functional roles in the cell and may have played a prominent part in metazoan evolution.
Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much ...supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.
Abstract
The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding ...genes. APPRIS selects a single protein isoform, the 'principal' isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants.
Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce ...dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level. We interrogate peptides from eight large-scale human proteomics experiments and databases and find that there is a single dominant protein isoform, irrespective of tissue or cell type, for the vast majority of the protein-coding genes in these experiments, in partial agreement with the conclusions from the most recent large-scale RNAseq study. Remarkably, the dominant isoforms from the experimental proteomics analyses coincided overwhelmingly with the reference isoforms selected by two completely orthogonal sources, the consensus coding sequence variants, which are agreed upon by separate manual genome curation teams, and the principal isoforms from the APPRIS database, predicted automatically from the conservation of protein sequence, structure, and function.
Abstract Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one ...source, 5′ untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends. Almost all of these N-terminal extensions are from highly abundant genes, which suggests that the novel regions we detect are just the tip of the iceberg. These upstream regions have characteristics that are not typical of coding exons. Their GC-content is remarkably high, even higher than 5′ regions in other genes, and a large majority have non-canonical start codons. Although some novel upstream regions have cross-species conservation - five have orthologues in invertebrates for example - the reading frames of two thirds are not conserved beyond simians. These non-conserved regions also have no evidence of purifying selection, which suggests that much of this translation is not functional. In addition, non-conserved upstream regions have significantly more peptides in cancer cell lines than would be expected, a strong indication that an aberrant or noisy translation initiation process may play an important role in translation from upstream regions.
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