REGULATION OF THE NONSENSE‐MEDIATED mRNA DECAY PATHWAY IN YEAST

Abstract

Nonsense-mediated mRNA decay (NMD) is a RNA surveillance pathway, which degrades transcripts harboring premature termination codons (PTCs). Upf1, a very well conserved protein essential for NMD, is phosphorylated in higher eukaryotes. The cycles of phosphorylation and dephosphorylation of Upf1p appear to be crucial for NMD, as blockade of either event in C. elegans and mammals prevents NMD. Here, we used in vitro and in vivo biochemical assays to show that S. cerevisiae Upf1p is a phosphoprotein. In vivo labeling experiments show that Upf1p is indeed phosphorylated in yeast. The phosphorylation of Upf1p in C. elegans is mediated by SMG-1, a PI3K related protein kinase. Sequence analyses were performed to identify putative proteins with homology to PI3-K-related kinases in yeast. The TOR1 gene was identified as a potential candidate for the yeast orthologue of SMG-1. We monitored the decay rates of several wild type and nonsense-containing mRNAs in TOR1 mutants. These studies suggest that the involvement of Tor1p in mRNA turnover is transcript-specific and includes both, PTC-containing transcripts, and mRNAs that are degraded via the deadenylation-dependent pathway. Ongoing experiments are focused on the phosphorylation status of Upf1p in tor1- cells. All together, these studies should provide more insights into the regulation of NMD surveillance mechanism by the phosphorylation of the Upf1 protein. This work is supported by grants from the NIH to C.I.G. (GM008102-3052, KO1 HL-04355-05, U54 CA96297-03). E.N.R. is supported by a graduate fellowship from PR-AABRE (NIH Grant Number P20 RR-016470).