Poster abstracts

Poster number 153 submitted by Toby Frazer

Dissecting unexpected connections between transcriptional control and nonsense mediated mRNA decay

Rene Arvola (Ohio State University Department of Molecular Genetics), Toby Frazer (Ohio State University Department of Molecular Genetics), Michael Schiff (Ohio State University Department of Physics), Debadrita Paul, Ryan Denniston, Rabab Abu Alhasan (Ohio State University Department of Molecular Genetics), Ralf Bundschuh (Ohio State University Department of Physics), Sharon Amacher, Guramrit Singh (Ohio State University Department of Molecular Genetics)

Abstract:
Nonsense Mediated mRNA Decay (NMD) degrades both aberrant transcripts containing Premature Termination Codons (PTCs) and “normal” transcripts with features that mimic PTCs. Regulation by NMD is pervasive and estimated to impact around 10% of the human transcriptome. In addition to “core” NMD factors such as UPF1, NMD is enhanced by the presence of the Exon Junction Complex (EJC) downstream of a stop codon, as is the case in mRNAs with PTCs or PTC-like contexts. UPF3 is a key NMD factor that engages with the EJC and the core NMD machinery to aid in PTC recognition. Many other enhancers of NMD have been identified through genetic screens, including ICE1, a member of the Little Elongation Complex (LEC) that regulates transcription by Pol II, though the mechanism is unclear. We sought to investigate the extent of LEC-mediated NMD regulation and found that depletion of ICE1 or other LEC component ELL1 in HCT116 human cell lines leads to global upregulation of PTC-containing mRNAs versus PTC-less, suggesting that ICE1’s previously implicated NMD role is performed within the LEC. Since ELL1 is shared with the related Super Elongation Complex (SEC) and multiple SEC components are highly ranked across genetic screens for NMD factors, we also investigated the SEC role in NMD. Depletion of SEC-specific ELL paralog ELL2 similarly led to upregulation of PTC-containing mRNAs. Moreover, knockdown of ICE1 and the SEC scaffold, AFF1 slowed decay of an NMD reporter. Immunoprecipitation of UPF3A and UPF3B identified multiple LEC and SEC components that interact with UPF3. Finally, depletion of LEC and SEC components led to reduced UPF3-EJC association, loss of UPF3 association with SR proteins, and changes in EJC deposition onto mRNA. In conclusion, we identify the SEC as a novel regulator of NMD that may act, in part through altering deposition of the EJC and NMD factors. Taken together, this work illuminates new connections between transcriptional control and mRNA decay processes.

References:
References:
[1] Baird TD, Cheng KC, Chen YC, Buehler E, Martin SE, Inglese J, and Hogg JR. Elife. 2018 Mar 12;7:e33178.

Keywords: mRNA, Transcription, Nonsense-Mediated Decay