Poster abstracts
Poster number 48 submitted by Caleb Embree
Pleiotropic Effects of Spliceosome Disruption Include Upregulation of Nonsense-Mediated mRNA Decay Targets
Caleb Embree (Department of Molecular Genetics, Center for RNA Biology, Ohio State University), Andreas Stephanou (Department of Molecular Genetics, Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, Ohio State University)
Abstract:
Pre-mRNA splicing, carried out by a large ribonucleoprotein machine known as the spliceosome, sculpts mRNAs by splicing out introns and decorating mRNA exon junctions with the exon junction complex (EJC). Both functions are critical for proper mRNA expression as they impact mRNA open reading frames and monitoring for premature translation termination by the nonsense mediated mRNA decay (NMD) pathway. Recently, genetic screens in human cell lines have identified spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from knockdowns of 18 different spliceosome components in K562 human cell lines, we have investigated the effect of spliceosome disruption on NMD efficiency by quantifying changes in abundance of NMD target and non-target mRNA isoforms. While we find that NMD targeted isoforms are upregulated when members of the catalytic spliceosome are knocked down, so are other non-canonical isoforms. After accounting for novel isoforms produced through missplicing, non-canonical isoforms remain upregulated compared to canonical isoforms, which are largely downregulated. Similarly, the canonical isoforms encoding NMD factors are downregulated , contrary to expectation, as NMD factor mRNAs are subjected to auto-regulatory feedback upon strong NMD inhibition. These results indicate that depletion of spliceosome components broadly changes the transcriptome, resulting in upregulation of NMD-targeted transcripts through mis-splicing or a minor reduction in NMD efficiency, or both. Interestingly, depletion of one spliceosomal component, CDC40, like the knockdown of EJC core protein EIF4A3, causes higher upregulation of NMD targeted isoforms as compared to other non-canonical isoforms, suggesting that its effects on NMD could be more direct. All together, we have shown that disruptions of pre-mRNA splicing have pleotropic effects on gene expression that also results in increased expression of NMD targeted transcripts. Although the precise reason of this effect on NMD targets remains unresolved, we have also observed increased abundance of NMD targeted transcripts in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Thus, altered levels of NMD regulated genes may contribute to the molecular phenotypes in spliceosomopathies.
Keywords: Splicing, NMD, mRNA Regulation