Recognition and degradation of mRNA by nonsense-mediated decay.

PROJECT SUMMARY/ABSTRACT
Gene expression is closely monitored to ensure fidelity in the conversion of genetic information into biological
activity. In eukaryotic cells a specialized quality control checkpoint exists during mRNA translation to evaluate
mRNA integrity and rapidly degrade transcripts containing a nonsense codon in their protein-coding region - a
signal that causes premature termination of translation and, if left unchecked, the accumulation of truncated
polypeptides. This pathway, referred to as nonsense-mediated mRNA decay (NMD), serves a vital biological
function by protecting cells from the deleterious consequences of incomplete proteins. In spite of over two
decades of study, fundamental questions central to this process remain poorly resolved, including how cells
distinguish normal and premature translation termination and what molecular events occur subsequently
between the translating mRNA and NMD machinery to incite accelerated degradation of the nonsense codon-
containing transcript. The long term objective of my research is to gain a comprehensive molecular
understanding of the events underlying the cell's ability to recognize and target nonsense-containing mRNA for
rapid decay.
In the last decade, we have made seminal discoveries in characterizing RNA features vital for the recognition
and efficient targeting of an mRNA to NMD. Recently, we have begun to dissect molecular events subsequent
to substrate recognition through identifying the complement of proteins specifically associated with nonsense-
containing mRNA and characterizing a catalytically-inactive mutant of the key NMD factor, UFP1. Notably, we
have described a functional interaction between the NMD machinery and prematurely terminating ribosomes
that exists in vivo and which is critical for dictating the fate of the aberrant transcript. We propose here to
capitalize on our past efforts and exploit novel tools and reagents to i) identify UPF1 RNA binding targets
globally to provide unprecedented insight into how NMD factor binding dictates the targeting of mRNA to NMD;
ii) interrogate further the functional interaction between prematurely terminating ribosomes and the NMD
machinery essential for targeting substrates to rapid decay; and iii) monitor NMD mRNP dynamics in vivo using
enzyme-catalyzed proximity labeling. Together, these studies will provide unprecedented insight into the
molecular events underlying an integral cellular RNA quality control pathway and regulator of gene expression.

Grant Number: 5R01GM143364-04
NIH Institute/Center: NIH
Principal Investigator: Kristian Baker