Friday, May 23, 2025
5/23/2025
Investigating UPF3 paralog function in Nonsense-Mediated mRNA Decay and Genetic Compensation - Abstract Nonsense Mediated mRNA Decay (NMD) degrades both aberrant transcripts containing Premature Termination Codons (PTCs) and “normal” transcripts with other specific features. Regulation by NMD is pervasive and estimated to impact around 10% of the transcriptome. UPF3 is a central NMD factor that bridges the mRNA bound Exon Junction Complex (EJC) with the rest of the NMD machinery and aids in PTC recognition. In mammals, there are two paralogs of UPF3, UPF3A and UPF3B, that have been documented to have both distinct biological roles and functions. We and others previously found that UPF3A can compensate for UPF3B in NMD, but is a weaker activator; moreover, this difference in activity is conferred by the “mid” domain. Moreover, overexpression of UPF3A, but not UPF3B stabilizes an NMD reporter mRNA. To understand the different propensities of UPF3 paralogs to stimulate NMD, we performed immunoprecipitation followed by mass spectrometry to identify their associated factors. In addition to EJC and NMD components, we identified transcriptional regulators and nucleocytoplasmic shuttling factors to be among some of the most enriched factors in UPF3A and UPF3B immunoprecipitation. We also identified members of the nuclear transcription regulating Little/Super Elongation Complex (LEC/SEC); the LEC was previously identified as an NMD factor that promotes UPF3B association with the EJC. UPF3 is a nucleocytoplasmic shuttling protein, but its nuclear functions are unknown. For the K99 phase (Aim 1), I am investigating the impact of these nuclear functions on UPF3 deposition and NMD activity. I hypothesize that nuclear import is required for UPF3 function in NMD, and the LEC/SEC mediate nuclear UPF3 deposition onto nascent mRNPs. I am also investigating the functional differences between the UPF3 paralogs in human cells and in zebrafish development; I hypothesize that differential nuclear deposition underlies some of the functional differences between UPF3A and UPF3B, and their distinct roles in vivo arise from these functional, rather than expression differences. This supplement is to help maintain research productivity of Aim 1 (K99 phase) of the parent grant after I return from maternity leave to allow me to focus on key aspects of research training. In zebrafish, Upf3a was implicated in the poorly characterized Genetic Compensation Response (GCR) via interaction with nuclear histone modifiers, and we found that this interaction is conserved in human cells. For the R00 phase (Aim 2), I will characterize the mechanism of GCR initiation from NMD-targeted mRNAs. This supplement will help facilitate a timely transition to an independent position and the research proposed in Aim 2. Taken together, this work will deepen our understanding of how events in the nucleus and cytoplasm are coordinated to regulate cytoplasmic mRNA decay and its feedback to transcriptional regulation.
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