Poster abstracts

Poster number 14 submitted by Caleb Embree

Disruption of the activated spliceosome impairs Nonsense Mediated mRNA Decay

Caleb Embree (Department of Molecular Genetics, Center for RNA Biology), Andreas Stephanou (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology)

Abstract:
Pre-mRNA splicing is tightly coupled to the downstream RNA quality control pathway nonsense mediated mRNA decay (NMD), both via deposition of the exon junction complex (EJC) and via production of alternatively spliced mRNAs that are cleared by NMD. Four screens for NMD factors in human cells have identified spliceosome components as potential contributors to NMD. Our analysis of these screens shows that the majority of spliceosome components identified are members of the activated spliceosome. Using spliceosome factor knockdown RNA-Seq datasets from the ENCODE project, we find that endogenous NMD targets are upregulated when several components of the catalytic spliceosome (e.g. AQR, SF3B1, EFTUD2) are knocked down. Conversely, knockdown of spliceosome components which leave before activation (e.g. SNRPC, SNRNP70, PRPF3) does not alter NMD. Notably, NMD is similarly disrupted in induced pluripotent stem cells derived from patients with Retinitis Pigmentosa (RP) caused by mutations in PRPF31, suggesting that the impact of spliceosome mutations on NMD needs to be considered to fully understand disease phenotype in RP and cranio-facial disorders caused by mutations in spliceosome components. As expected, depletion of spliceosomal proteins leads to widespread mis-splicing, with a large fraction of genes producing novel splicing patterns as well as novel isoforms. Interestingly, concentrations of novel isoforms that are NMD targets represent a small fraction of overall NMD burden, thereby disputing the earlier proposal that spliceosome disruption overwhelms NMD via production of excess novel targets. We find that in several spliceosome knockdowns, levels of canonical mRNAs of NMD factor genes (ex. UPF3A and UPF3B), are downregulated, which could be one possible explanation for lowered NMD efficiency. Another hypothesis under investigation is compromised EJC deposition upon reduced function of the catalytic spliceosome. Overall, our work reveals a much broader impact of the spliceosome on mRNA quality control processes in human cells.

Keywords: Splicing, NMD, mRNA Regulation