Gene expression is regulated on numerous levels including transcription, RNA processing, translation, and RNA degradation. Messenger RNA (mRNA) is surveilled during translation by at least three quality control mechanisms committed to the elimination of defective mRNAs;non-sense mediated decay (NMD), no-go decay (NGD), and non-stop decay (NSD). NSD targets transcripts lacking stop codons, which not only generate aberrant proteins, but also stall ribosomes, leading to an overall decrease in translation efficiency. The mechanism of NSD in eukaryotes is not well understood. NSD is presumably triggered by the stalling of ribosomes on non-stop mRNA transcripts. In S. cerevisiae the non-stop mRNA is degraded either when the exosome-associated factor Ski7 interacts with stalled ribosomes to trigger 3'to 5'decay, or in a second pathway in which the mRNA is decapped and degraded in a 5'to 3'manner by nucleases. When eukaryotic ribosomes translate non-stop mRNA, translation presumably proceeds through the poly(A) tail creating protein products with poly-lysine C-termini that can disrupt function. These aberrant poly-Lys proteins are thought to be targeted by specific machinery. Recent studies of ?ltn knockouts in S. cerevisiae suggest that non-stop proteins may be tagged for degradation by the ribosome-associated ubiquitin ligase Ltn1. However, the precise contribution of Ltn1 to NSD is unknown. The goals of the work proposed here are to further delineate the mechanisms of mRNA the eukaryotic quality control pathway non-stop decay by studying the mechanism and targets of non-stop decay in S. cerevisiae. Specifically, these studies will investigate the targets and fundamental mechanism of NSD in eukaryotes using methods developed in the Green lab to measure the rates of peptide elongation, peptide release and ribosome release during in vitro translation. Mass spectrometry based ubiquitination assays and new deep-sequencing technologies (ribosome profiling) will be employed in addition to standard biochemical assays.
The aim of these studies is to address how translation of non-stop mNRA impacts the ribosome, investigate the precise contribution of Ltn1 to NSD, and identify mRNAs targeted for NSD. This study will also address the possibility that NSD, like NMD, not only targets defective mRNAs, but also performs a broader function in the cell by regulating a subset of mRNA transcripts in order to control gene expression levels. This work will reveal the fundamental mechanism by which mRNAs lacking a stop-codon are decayed by NSD in eukaryotes and identify the in vivo of NSD targets in eukaryotes, which could provide information relevant to human health and disease.

Public Health Relevance

Errors in mRNA processing have deleterious effects and are associated with a variety of human diseases, including ? - thalassemia and certain cancers. The precise mechanism and targets of non-stop decay (NSD), an mRNA quality control mechanism committed to the elimination of defective mRNAs during translation, are unknown. The work proposed here will characterize the mechanism of eukaryotic NSD and identify the full panel of NSD targets, which could provide information relevant to design of therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM100608-01
Application #
8254541
Study Section
Special Emphasis Panel (ZRG1-F04B-D (20))
Program Officer
Carter, Anthony D
Project Start
2012-04-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$53,942
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Koutmou, Kristin S; McDonald, Megan E; Brunelle, Julie L et al. (2014) RF3:GTP promotes rapid dissociation of the class 1 termination factor. RNA 20:609-20