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Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity.

ABSTRACT: Alternative splicing dramatically increases transcriptome complexity but its contribution to proteome diversity remains controversial. Exon-exon junction spanning peptides provide direct evidence for the translation of specific splice isoforms and are critical for delineating protein isoform complexity. Here we found that junction-spanning peptides are underrepresented in publicly available mass spectrometry-based shotgun proteomics data sets. Further analysis showed that evolutionarily conserved preferential nucleotide usage at exon boundaries increases the occurrence of lysine- and arginine-coding triplets at the end of exons. Because both lysine and arginine residues are cleavage sites of trypsin, the nearly exclusive use of trypsin as the protein digestion enzyme in shotgun proteomic analyses hinders the detection of junction-spanning peptides. To study the impact of enzyme selection on splice junction detectability, we performed in-silico digestion of the human proteome using six proteases. The six enzymes created a total of 161,125 detectable junctions, and only 1,029 were common across all enzyme digestions. Chymotrypsin digestion provided the largest number of detectable junctions. Our experimental results further showed that combination of a chymotrypsin-based human proteome analysis with a trypsin-based analysis increased detection of junction-spanning peptides by 37% over the trypsin-only analysis and identified over a thousand junctions that were undetectable in fully tryptic digests. Our study demonstrates that detection of proteome diversity resulted from alternative splicing is limited by trypsin cleavage specificity, and that complementary digestion schemes will be essential to comprehensively analyze the translation of alternative splicing isoforms.

SUBMITTER: Wang X

PROVIDER: S-EPMC5836368 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity.

Wang Xiaojing X Codreanu Simona G SG Wen Bo B Li Kai K Chambers Matthew C MC Liebler Daniel C DC Zhang Bing B

Molecular & cellular proteomics : MCP 20171208 3

Alternative splicing dramatically increases transcriptome complexity but its contribution to proteome diversity remains controversial. Exon-exon junction spanning peptides provide direct evidence for the translation of specific splice isoforms and are critical for delineating protein isoform complexity. Here we found that junction-spanning peptides are underrepresented in publicly available mass spectrometry-based shotgun proteomics data sets. Further analysis showed that evolutionarily conserve ...[more]

PMID: 29222161

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Project description:BACKGROUND: Alternative splicing (AS) is a widespread phenomenon in higher eukaryotes but the extent to which it leads to functional protein isoforms and to proteome expansion at large is still a matter of debate. In contrast to animal species, for which AS has been studied extensively at the protein and functional level, protein-centered studies of AS in plant species are scarce. Here we investigate the functional impact of AS in dicot and monocot plant species using a comparative approach. RESULTS: Detailed comparison of AS events in alternative spliced orthologs from the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice) revealed that the vast majority of AS events in both species do not result from functional conservation. Transcript isoforms that are putative targets for the nonsense-mediated decay (NMD) pathway are as likely to contain conserved AS events as isoforms that are translated into proteins. Similar results were obtained when the same comparison was performed between the two more closely related monocot species rice and Zea mays (maize).Genome-wide computational analysis of functional protein domains encoded in alternatively and constitutively spliced genes revealed that only the RNA recognition motif (RRM) is overrepresented in alternatively spliced genes in all species analyzed. In contrast, three domain types were overrepresented in constitutively spliced genes. AS events were found to be less frequent within than outside predicted protein domains and no domain type was found to be enriched with AS introns. Analysis of AS events that result in the removal of complete protein domains revealed that only a small number of domain types is spliced-out in all species analyzed. Finally, in a substantial fraction of cases where a domain is completely removed, this domain appeared to be a unit of a tandem repeat. CONCLUSION: The results from the ortholog comparisons suggest that the ability of a gene to produce more than one functional protein through AS does not persist during evolution. Cross-species comparison of the results of the protein-domain oriented analyses indicates little correspondence between the analyzed species. Based on the premise that functional genetic features are most likely to be conserved during evolution, we conclude that AS has only a limited role in functional expansion of the proteome in plants.

Dataset Information

Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity.

Publications

Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity.

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