Impact of alternative splicing on the human proteome
ABSTRACT: 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 dynamic perturbations in mRNA splicing patterns that follow depletion of the core spliceosome factor PRPF8 alter the composition of the proteome. We integrate RNA-sequencing (to comprehensively report intron retention, differential transcript usage and gene expression) with a newly developed data-independent acquisition (DIA) method, SWATH-MS (Sequential Window Acquisition of all THeoretical spectra – mass spectrometry), to capture an unbiased and quantitative snapshot of constitutive and alternative splicing events at the protein level. Whereas intron retention is accompanied by decreased protein abundance, dynamic alterations in differential transcript usage and gene expression alter protein abundance proportionally to transcript levels. Our findings exemplify how RNA splicing exerts a pervasive effect linking isoform expression in the human transcriptome with proteomic diversity, and provide a toolkit for studying its perturbation in human diseases.
Project description:The human plasma proteome is clinically highly significant and has been studied extensively because it is thought that its molecular makeup provides a window into the health state of an individual. However, neither the quantitative variability of the plasma proteome nor the origins of the variation are known. To determine the relative contributions of heritability, environmental and longitudinal factors to plasma proteome variability we systematically decomposed the biological variance of 1904 peptides defining 342 unique plasma protein profiles from 232 plasma samples that were collected with 2-7 year intervals from monozygotic (MZ) and dizygotic (DZ) twins. The data were collected via SWATH mass spectrometry (SWATH-MS), an emerging technology characterized by high degree of reproducibility and quantitative accuracy. The data indicate abundance variability is an important feature for different proteins among population, that the abundances of about 20% of plasma protein are considerably heritable, and that the degrees of genetic control and aging effects vary across specific biological processes. Moreover, we identified 13 cis- SNPs significantly influencing the abundance level of specific plasma proteins. These results substantially extend the understanding of the impact of heritability on the human proteomic dynamics and therefore have implications for the effective design of plasma-based biomarker studies.
Project description:Human Trisomy 21 (T21), which causes Down Syndrome (DS), is the most common known cause of intellectual disability. However, the molecular basis for DS phenotypic variability remains poorly understood. Here we used SWATH mass spectrometry (SWATH-MS) to quantify protein abundance and protein turnover in fibroblasts from a monozygotic twin pair discordant for T21, and to profile protein expression in 11 unrelated DS individuals and age-matched controls. The integration of the steady state and turnover proteomic data sets with transcript profiles indicated that protein-specific degradation of members of stoichiometric complexes presents a major determinant of T21 gene dosage outcome, a primary effect that was not apparent from genomic data. The data also reveal that T21 results in extensive proteome remodeling similarly affecting proteins encoded by all chromosomes. Finally, we found broad, organelle-specific posttranscriptional effects such as significant down-regulation of the mitochondrial proteome contributing DS hallmarks and variability.
Project description:This ArrayExpress record contains meta-data and results of quantitative analysis of cell lines from the NCI-60 panel using pressure cycling technology (PCT) and SWATH-mass spectrometry. Each cell line was analyzed in duplicate. Raw data files are available at the EMBL-EBI protemics data archive (PRIDE) at accession PXD003539 (http://www.ebi.ac.uk/pride/archive/projects/PXD003539). Since the record here does not include the raw data files and hence there is no need to explicitly link individual replicate to a raw file, each sample is only listed once in the ArrayExpress samples table for clarity.
Project description:Galiellalactone (GL) is a fungal metabolite that presents antitumor and anti-inflammatory activities in vitro and in vivo. Previous studies have shown that GL targets NF-KB and STAT3 pathways and induces G2/M cell cycle arrest in androgen-insensitive prostate cancer cells. In this study, we show that GL-induced cell cycle arrest is independent of the NF-KB and STAT3 pathways in DU145 and PC-3 cells, and also that GL did not affect cell cycling in androgen-sensitive prostate cancer cell such as LNCaP and 22Rv1 cells. In addition, we showed confluence resistance to GL in DU145 cells. Using a SWATH proteomic approach we identified the down-regulation of Nucleolar and spindle associated protein 1 (NUSAP1) under DU145 confluence and in LNCaP cells. Also, the inhibition of NUSAP1 by siRNAs induced resistance to GL in DU145 cells, suggesting that NUSAP1 may be a target for GL and could be useful as biomarker for responsiveness of the antitumor activity of GL.
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. Small-RNA libraries from two control samples and four knockdowns in germline or somatic tissues of the Drosophila melanogaster ovary.
Project description:To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. 87% of introns assayed manifest more than 50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly, or slowly, with ~3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns and introns annotated as alternative. FinallyFinally, S2 cells expressing the slow RpII215C4 mutant manifest substantially less intron retention than wild-type S2 cells. Examination of Total pA and Nascent RNA from 2 different cell populations and isolated fly heads.
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes aberrant transcripts arising from mutations and transcriptional mistakes. Moreover, differential transcript processing such as alternative precursor mRNA splicing (AS) can generate NMD substrates, however, the extent of coupled AS and NMD remained unclear. To investigate NMD targeting of AS variants, we performed transcriptome-wide splicing studies using Arabidopsis thaliana single and double mutants in the NMD factor homologues UPF1 and UPF3, as well as samples treated with the translation inhibitor cycloheximide (CHX). Our analyses revealed that at least 17.5% of all multi-exon, protein-coding genes produce splicing variants of all major types that are targeted by NMD, with a significant fraction originating from the splicing of cryptic introns. Furthermore, accumulation of many intron-retained mRNAs in the mutants, but not in response to CHX suggests the action of distinct routes of NMD with variable impact of translation. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. NMD-dependent AS variants are linked to diverse biological functions, including the poison exon-mediated regulation of signaling and posttranslational protein modification components. In addition to mRNAs, numerous non-coding RNAs as well as newly identified transcripts derived from intergenic regions were shown to be NMD responsive. In summary, our comprehensive analysis of AS-coupled NMD provides strong evidence for a major function of this pathway in shaping the transcriptome, having fundamental implications in gene regulation and quality control of transcript processing steps in higher eukaryotes. Comparison of gene expression and alternative splicing patterns in control and nonsense-mediated decay (NMD)-impaired Arabidopsis seedlings
Project description:We collected 14 stock Hela aliquots from 13 different laboratories across the globe and cultured them in the same conditions. We extensively profiled the genome-wide copy numbers, mRNAs, proteins, protein turnover rates by genomics techniques and the highly reproducible and accurate proteomic method, SWATH mass spectrometry. The cell lines were also phenotyped with respect to the ability of transfected Let7 mimics to modulate Salmonella infection. We discovered significant heterogeneity between Hela variants especially differences between the CCL2 and Kyoto lines collected from different sites. In addition, we observed progressive divergence within a specific cell line over 50 successive passages. By associating proteotype and phenotype we identified molecular patterns that varied between cell lines and explained varying responses to Salmonella infection across the cells. The results furthermore quantify how the cells respond to genomic variability across the transcriptome and proteome.
Project description:A better understanding of the mechanisms for plant in response to abiotic stresses is key for the improvement of plant to resistant to the stresses. Much has been known for the regulation of gene expression in response to salt stress at transcriptional level, however, little is known at posttranscriptional level for this response. Recently, we identified that SKIP is a component of spliceosome and is necessary for the regulation of alternative splicing and mRNA maturation of clock genes. In this study, we observed that skip-1 is hypersensitive to salt stress. SKIP is necessary for the alternative splicing and mRNA maturation of several salt tolerance genes, e.g. NHX1, CBL1, P5CS1, RCI2A, and PAT10. Genome-wide analysis reveals that SKIP mediates the alternative splicing of many genes under salt stress condition, most of the new alternative splicing events in skip-1 is intron retention, which leads to the premature termination codon in their mRNA. SKIP also controls the alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt stress condition. Therefore, this study addresses a fundamental question on how the mRNA splicing machinery contributes to salt response at a posttranscriptional level. Totally six samples, two treatments and two genotypes, and each have two replicats.
Project description:Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and primarily enzymatically detoxified by the mitochondrial ß-cyanoalanine synthase (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis results in physiological alterations in the plant that suggest that the function of HCN is a gasotransmitter molecule. Label-free quantitative proteomic analysis of enriched mitochondrial samples isolated from the wild type and cas-c1 mutant revealed significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins that are only present or more abundant in the mutant background. Gene ontology classification of these proteins highlights proteomic changes that explains the root hairless phenotype and the altered immune response observed in the cas-c1 mutant. The mechanism of action of cyanide as a signaling molecule has been addressed using two proteomic approaches focused on identifying the S-cyanylation of cysteine as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and the direct untargeted analysis of proteins using LC-MS/MS identified a set of 163 proteins susceptible to S-cyanylation that included sedoheptulose 1, 7-bisphosphatase (SBPase), the peptidyl-prolyl cis-trans isomerase (CYP20-3) and enolase 2 (ENO2). In vitro analysis of these proteins identified that this modification in the SBPase Cys74, CYP20-3 Cys259 and ENO2 Cys346 residues affected the enzymatic activity of the enzymes. GO classification and protein-protein interaction cluster analysis revealed the function of S-cyanylation in the regulation of primary metabolic pathways, such as glycolysis, and the Calvin and S-adenosylmethionine cycles.