Poster abstracts

Poster number 38 submitted by Debadrita Paul

Investigating the unexpected connection between promoter proximal RNA Pol II pausing and nonsense-mediated mRNA decay

Debadrita Paul (Molecular, Cellular and Developmental Biology Program), Rene Arvola (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)

Abstract:
mRNA biogenesis in the nucleus has a profound impact on transcript fate in the cytoplasm. For example, co-transcriptional deposition of the exon junction complex (EJC) enhances nonsense-mediated mRNA decay (NMD) in the cytoplasm. NMD eliminates transcripts with premature termination codons and regulates ~10% of the transcriptome. We have identified an unexpected connection between NMD and RNA Polymerase II (Pol II) promoter-proximal pausing, which creates an optimal window for RNA processing. Proteomic studies reveal interactions between core NMD factor UPF3 and transcriptional super elongation complex (SEC) and little elongation complex (LEC). SEC competes with LEC and mediates release of Pol II from promoter proximal pausing into elongation. We find that depletion of pause release factors (SEC) further downregulates NMD targets, while depletion of pause-stabilizing factors (LEC) has the opposite effect. In our first aim, we will determine transcriptome-wide decay rates of NMD target intermediates under conditions that stabilize or release Pol II pausing. To this end, we have optimized a 4-thiouridine based pulse-labeling assay to differentiate between altered RNA decay vs transcription. Our second aim is to identify the molecular basis of NMD enhancement by promoter-proximal pausing. We hypothesize that Pol II pausing promotes EJC deposition to enhance NMD. We will measure RNA-dependent EJC occupancy in promoter proximal regions via ChIP-Seq under conditions that stabilize (SEC knockdown) or release (LEC knockdown) paused Pol II. To determine how EJCs are recruited to nascent transcripts, we will similarly test EJC occupancy upon knockdown of (i) subunits of transcription/export (TREX) complex, which associates with nascent transcripts as well as Pol II and EJC subunit EIF4A3, and (ii) Spt6, a pause release factor that we have identified as an EJC interactor. Our work will reveal how Pol II pausing preprograms mRNA fate through splicing-independent EJC deposition.

References:
Watson, M. J., & Thoreen, C. C. (2022). Measuring mRNA decay with roadblock‐qPCR. Current protocols, 2(1), e344.
Aoi, Y., Shah, A. P., Ganesan, S., Soliman, S. H., Cho, B. K., Goo, Y. A., ... & Shilatifard, A. (2022). SPT6 functions in transcriptional pause/release via PAF1C recruitment. Molecular cell, 82(18), 3412-3423.
Viphakone, N., Sudbery, I., Griffith, L., Heath, C. G., Sims, D., & Wilson, S. A. (2019). Co-transcriptional loading of RNA export factors shapes the human transcriptome. Molecular Cell, 75(2), 310-323.
Mabin, J. W., Woodward, L. A., Patton, R. D., Yi, Z., Jia, M., Wysocki, V. H., ... & Singh, G. (2018). The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity. Cell reports, 25(9), 2431-2446.

Keywords: NMD, Promoter proximal pausing, EJC