Protein diversity exceeds gene count due to alternative splicing and post-translational modifications
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Peer-reviewed literature establishes that alternative splicing allows cells to generate protein diversity from limited gene counts, but the combined quantitative relationship involving post-translational modifications is only partially covered.
Abstract Alternative splicing of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversity from a limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.
Figure 3: Interplay between splicing and epigenetic modifications in spermatogenesis and regulatory feedback loops involving splicing in T cell activation. Similar content being viewed by others The physiology of alternative splicing Article 13 October 2022 The regulation and function of post-transcriptional RNA splicing Article 11 April 2025 Alternative splicing is coupled to gene expression in a subset of variably expressed genes Article Open access 04 November 2024 Explore related subjects Discover the latest articles and news in related subjects. Alternative splicing RNA splicing Development Differentiation Organogenesis References Pan, Q., Shai, O., Lee, L. J., Frey, B. J.
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RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat. Med. 18 , 766–773 (2012). CAS PubMed PubMed Central Google Scholar Refaat, M. M. et al. Genetic variation in the alternative splicing regulator RBM20 is associated with dilated cardiomyopathy. Heart Rhythm 9 , 390–396 (2012). PubMed Google Scholar Brauch, K. M. et al. Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy. J. Am. Coll. Cardiol. 54 , 930–941 (2009). CAS PubMed PubMed Central Google Scholar Beqqali, A. et al.
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Ethics declarations Competing interests The authors declare no competing financial interests. Supplementary information Supplementary information S1 (table) (download PDF ) RNA-binding proteins (RBPs) regulating alternative splicing in developmental or differentiation contexts (PDF 156 kb) PowerPoint slides PowerPoint slide for Fig. 1 (download PPT ) PowerPoint slide for Fig. 2 (download PPT ) PowerPoint slide for Fig.
The human transcriptome is composed of a vast RNA population that undergoes further diversification by splicing. Detecting specific splice sites in this large sequence pool is the responsibility of the major and minor spliceosomes in collaboration with numerous splicing factors. This complexity makes splicing susceptible to sequence polymorphisms and deleterious mutations. Indeed, RNA mis-splicing underlies a growing number of human diseases with substantial societal consequences. Here, we provide an overview of RNA splicing mechanisms followed by a discussion of disease-associated errors, with an emphasis on recently described mutations that have provided new insights into splicing regulation. We also discuss emerging strategies for splicing-modulating therapy.
Abstract
Post-translational modifications (PTMs) and splicing are important regulatory processes for controlling protein function and activity. Despite examples of interplay between alternative splicing and cell signaling in literature, there have been few detailed analyses of the impacts of alternative splicing on PTMs, partly due to difficulties in extracting PTM information from splicing measurements. We developed a computational pipeline, PTM Projection Onto Splice Events (PTM-POSE), to identify “prospective” PTM sites in alternative isoforms and splice events recorded in databases using only the genomic coordinates of a splice event or isoform of interest. Importantly, PTM-POSE integrates various PTM-specific databases and tools to allow for deeper analysis of the individual and global impact of spliced PTMs on isoform function, protein interactions, and regulation by enzymes like kinases. Using PTM-POSE, we performed a systematic analysis of PTM diversification across isoforms annotated in the Ensembl database. We found that 32% of PTMs are excluded from at least one Ensembl isoform, with palmitoylation being most likely to be excluded (49%) and glycosylation and crotonylation exhibiting the highest constitutive rates (75% and 94%, respectively). Further, approximately 2% of prospective PTM sites exhibited altered regulatory sequences surrounding the modification site, suggesting that regulatory or binding interactions might be different in these proteoforms. When comparing splicing of phosphorylation sites to measured phosphorylation abundance in KRAS-expressing lung cells, differential inclusion of phosphorylation sites correlated with phosphorylation levels, particularly for larger changes in inclusion (
>
20%). To better understand how splicing diversification of PTMs may alter protein function and regulatory networks in specific biological contexts, we applied PTM-POSE to exon utilization measurements from TCGASpliceSeq of prostate tumor samples from The Cancer Genome Atlas (TCGA) and identified 1,489 PTMs impacted by ESRP1-correlated splicing, a splicing factor associated with worsened prognosis. We identified protein interaction and regulatory networks that may be rewired as a result of differential inclusion of PTM sites in ribosomal and cytoskeletal proteins. We also found instances in which ESRP1-mediated splicing impacted PTMs by altering flanking residues surrounding specific phosphorylation sites that may be targets of 14-3-3 proteins and SH2 domains. In addition, SGK1 signaling was found to be influenced by ESRP1 expression through increased inclusion of SGK1 substrates in ESRP1-expressing patients. Based on validation in a separate prostate cancer cohort from the Chinese Prostate Cancer Genome and EpiGenome Atlas (CPGEA), this correlated with increased phosphorylation of SGK1 substrates, particularly when SGK1 was predicted to be active. From this work, we highlighted the extensive splicing-control of PTM sites across the transcriptome and the novel information that can be gained through inclusion of PTMs in the analysis of alternative splicing. Importantly, we have provided a publicly available python package (PTM-POSE:
https://github.com/NaegleLab/PTM-POSE
) and all associated data for use by the broader scientific community to allow for continued exploration of the relationship between splicing and PTMs.
Protein-protein interactions play an essential role in nearly all biological processes, and it has become increasingly clear that in order to better understand the fundamental processes that underlie disease, we must develop a strong understanding of both their context specificity (e.g., tissue-specificity) as well as their dynamic nature (e.g., how they respond to environmental changes). While network-based approaches have found much initial success in the application of protein-protein interactions (PPIs) towards systems-level explorations of biology, they often overlook the fact that large numbers of proteins undergo alternative splicing. Alternative splicing has not only been shown to diversify protein function through the generation of multiple protein isoforms, but also remodel PPIs and affect a wide range diseases, including cancer. Isoform-specific interactions are not well characterized, so we develop a computational approach that uses domain-domain interactions in concert with differential exon usage data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression project (GTEx). Using this approach, we can characterize PPIs likely disrupted or possibly even increased due to splicing events for individual TCGA cancer patient samples relative to a matched GTEx normal tissue background.
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