Talk on Friday 01:36-01:48pm submitted by DaJuan Whiteside
Mechanistic insight into the association of the NMD factor, Upf1p, with mRNA in Saccharomyces cerevisiae
DaJuan L. Whiteside (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control pathway that identifies and targets aberrant mRNAs for accelerated degradation. The rapid turnover of nonsense-containing mRNAs promotes fidelity in gene expression by preventing the accumulation of truncated polypeptides that could have deleterious effects to the cell. While three core proteins are required for NMD in yeast - namely Upf1p, Upf2p, and Upf3p - Upf1p is the only factor with known enzymatic activity and an ability to bind RNA directly. Key mechanistic events underlying NMD substrate recognition currently remain unclear, including the dependence of Upf1p binding on mRNA translation and how specifically the NMD machinery is recruited to target substrates.
We have developed a biochemical assay involving RNA immunoprecipitation (RIP) coupled with quantitative RT-PCR to evaluate mRNA features and protein requirements for Upf1p binding in the yeast Saccharomyces cerevisiae. Specifically, chromosomally expressed, epitope-tagged Upf1p is immunoprecipitated from yeast cell lysates and associated RNA quantified by real-time PCR. Taqman probes specific to endogenous or plasmid-based mRNA reporters allow for multiplex PCR reactions and the evaluation of multiple RNAs in a single sample.
Our studies have revealed the following: (i) Upf1p preferentially binds nonsense-containing mRNA, (ii) Upf1p binding to RNA is not dependent on the other NMD factors Upf2p or Upf3p, (iii) translation enhances, but is not absolutely required for Upf1p binding, (iv) Upf1p displays a length-dependent association with nonsense-containing mRNAs and, lastly, (v) secondary structure downstream of a premature termination event can stabilize NMD substrates and modify Upf1p association with the mRNA. Additionally, analysis of previously characterized mutant alleles of Upf1p reveal that neither the ATP binding or hydrolysis activity of Upf1p is required for its binding to RNA. Taken together, our data provide insight into the mechanism of Upf1p association with mRNA and NMD substrate recognition in yeast.
Keywords: mRNA decay