Abstract
SummaryAlternative 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.
Highlights
Next-generation RNA sequencing (RNA-seq) has identified alternative splicing of RNA transcripts as a key mechanism that may underlie the diversification of proteins encoded in the human genome
Alternative splicing is a critical determinant of genome complexity and, by implication, is assumed to engender proteomic diversity
We have developed an integrative approach to ask whether perturbations in mRNA splicing patterns alter the composition of the proteome
Summary
Next-generation RNA sequencing (RNA-seq) has identified alternative splicing of RNA transcripts as a key mechanism that may underlie the diversification of proteins encoded in the human genome. More recent studies have incorporated expression data, such as evidence from RNA-seq experiments, in the interrogation of proteomic datasets to reduce mapping noise (Lopez-Casado et al, 2012; Ning and Nesvizhskii, 2010; Sheynkman et al, 2013; Tanner et al, 2007) None of these studies have attempted to quantify the contribution of alternative splicing to proteomic diversity in a systematic manner. We seek to address this fundamental biological question by asking whether selective perturbations in RNA splicing patterns manifest as changes in the composition of the proteome By using this system, we have established in a quantitative manner how changes in splicing of a subset of transcripts determine differential protein expression
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