Bradley, R. K. | bioRxiv Science

bioRxiv2018

Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD

DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). While DUX4s transcriptional activity has been extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-induced pathology.

genomics↗

bioRxiv2018

RNA components of the spliceosome regulate tissue- and cancer-specific alternative splicing

Alternative splicing of pre-mRNAs plays a pivotal role during the establishment and maintenance of human cell types. Characterizing the trans-acting regulatory proteins that control alternative splicing in both healthy and malignant cells has therefore been the focus of much research. Recent work has established that even core protein components of the spliceosome, which are required for splicing to proceed, can nonetheless contribute to splicing regulation by modulating splice site choice. We here demonstrate that the RNA components of the spliceosome likewise influence alternative splicing decisions and contribute to the establishment of global splicing programs. Although these small nuclear RNAs (snRNAs), termed U1, U2, U4, U5, and U6 snRNA, are present in equal stoichiometry within the spliceosome, we found that their relative levels vary by an order of magnitude during development, across tissues, and between normal and malignant cells. Physiologically relevant perturbation of individual snRNAs drove widespread gene-specific differences in alternative splicing, but not transcriptome-wide splicing failure. Genes that were particularly sensitive to variations in snRNA abundance in a breast cancer cell line model were likewise preferentially mis-spliced within a clinically diverse cohort of invasive breast ductal carcinomas. As aberrant mRNA splicing is prevalent in many solid and liquid tumors, we propose that a full understanding of dysregulated pre-mRNA processing in cancers requires study of the RNA as well as protein components of the splicing machinery.

genomics↗