Integrative multiomic analysis reveals co-ordinated alternative splicing in human bone marrow stromal stem cells

Alternative splicing is essential for the production of diverse messenger RNAs from a single gene. However our understanding of alternative splicing and its regulation during processes such as osteoblast differentiation remains incomplete. Using differentiating hMSC-TERT4 cells as a model, we performed deep short- and long-read RNA sequencing, revealing a highly integrated multiphasic differentiation program. Analysis for splicing revealed extensive changes during lineage commitment, in genes associated with known transcriptional regulators including RUNX2, TEAD1, CTNNB1 and NFATC4. During late-stage matrix osteoblast differentiation (maturation), splicing changes occurred in genes encoding cytoskeletal and extracellular matrix proteins, and Golgi and vesicle trafficking proteins. Analysis of proteomic and phosphoproteomic profiles confirmed the presence of alternative splicing in proteins participating in these biological processes as well as in RNA splicing and autophagy, with splicing involving a variety of protein domains, regions and PTM sites. Together, the transcriptomic and protein splicing landscapes provide a comprehensive description of how mesenchymal stromal cells progressively acquire an osteoblastic phenotype.