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 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, I will investigate 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 will also investigate 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. 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, I will characterize the mechanism of
GCR initiation from NMD-targeted mRNAs. I hypothesize UPF3A/Upf3a-stimulated NMD is required for GCR
and UPF3A/Upf3a presence at these genomic loci determines GCR. Importantly, I have assembled a strong
mentoring committee that will contribute their expertise to both my research training and professional
development. This work will elucidate how the nuclear functions of the UPF3 paralogs impact NMD and how
their functions contribute to developmental outcomes. 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.

Grant Number: 5K99GM154061-02
NIH Institute/Center: NIH
Principal Investigator: Rene Arvola