Poster abstracts
Poster number 46 submitted by Caleb Embree
Loss of Function of Activated Spliceosome Components Reduces Nonsense Mediated mRNA Decay Efficiency
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 macromolecular machine called the spliceosome, sculpts mRNA sequences for translation of genetic code into proteins. Sometimes, alternative splicing or spliceosome errors can disrupt mRNA open reading frames. Therefore, to muffle translation of proteins from such mRNAs, splicing is tightly linked to nonsense-mediated mRNA decay (NMD), which rapidly degrades mRNAs where translation terminates prematurely. Splicing and NMD are physically connected through the exon junction complex (EJC), which is deposited on mRNA by the spliceosome and is relied upon by the ribosome to identify premature translation termination.
Multiple genetic screens in human cells for NMD factors have identified spliceosome components as potential candidates. While components of all spliceosome subcomplexes were identified among the top hits in these screens, >50% of the spliceosome proteins detected are from the catalytic spliceosome. Using data from the ENCODE consortium, we are testing how NMD activity is altered in myelogenous leukemia K562 cell lines upon knockdown of spliceosome components. Depletion of activated spliceosome components (e.g., AQR, CDC40, and SF3B1) upregulates mRNAs subjected to alternative splicing-linked NMD (AS-NMD) when compared to non-target isoforms from the same genes. However, knockdown of spliceosome components only present in the pre-catalytic steps of pre-mRNA splicing (e.g., PRPF4, SNRNP70) does not upregulate AS-NMD targets. As expected, depletion of all spliceosome components tested results in altered splicing patterns and generation of novel mRNA isoforms. However, novel, NMD sensitive isoforms accumulate to a higher level in cells where spliceosome component knockdown impairs NMD. Together these data indicate that depletion of components of the activated spliceosome lowers global NMD efficiency. We are examining the hypotheses that reduced NMD efficiency results from either superfluous generation of NMD targets, or due to defects in EJC deposition, or both. Our results further illuminates the dynamic interplay between pre-mRNA splicing and NMD and suggests that in diseases caused by mutations in spliceosome components (e.g., myelodysplastic syndromes), reduced NMD efficiency also contributes to altered gene expression.
Keywords: NMD, pre-mRNA Splicing, mRNA Regulation