RUI: Regulation of Gene Expression by the Nonsense-mediated mRNA Decay Pathway of Yeast

Saint Olaf College, Northfield MN

Investigators

Abstract

0091300 Jeffrey Dahlseid Nonsense-mediated mRNA decay (NMD) accelerates the degradation of mRNAs that undergo premature translation termination due to a nonsense mutation. NMD exists in all eukaryotes thus far examined, from yeast to man, and safeguards cells against the accumulation of potentially deleterious protein fragments encoded by so-called nonsense mRNAs. In budding yeast, the UPF1, UPF2, and UPF3 genes are required for NMD. These genes also affect the accumulation of hundreds of wild-type mRNAs, which suggests that NMD is an important part of the natural cellular repertoire for regulating wild-type gene expression. The primary objective of this research is to study the role of NMD in regulating the expression of wild-type genes. NMD affects the mRNA accumulation of several wild-type genes that encode proteins involved in chromosome transmission and stability. The mRNAs for CTF13, which encodes an essential protein of the CBF3 kinetochore complex, two additional kinetochore proteins, and five proteins that affect telomere function are elevated in upf mutant yeast strains. The three specific aims of this research are to determine if NMD directly affects the stability of these mRNAs, to characterize the recognition and degradation of any that are wild-type mRNA targets of NMD, and to investigate potential coordinate regulation of wild-type gene expression by NMD. Analysis of expression from promoter-reporter gene fusions and measurement of mRNA decay and transcription rates will be used to determine whether NMD directly affects mRNA stability or exerts indirect influence upon mRNA transcription. Decay of many wild-type yeast mRNAs involves deadenylation followed by decapping and 5'->3' exonucleolysis, whereas nonsense mRNAs bypass deadenylation but then undergo the same decay. Analysis of deadenylation rates and susceptibility to decapping and 5'->3' exonucleolysis will be used to determine whether NMD degrades wild-type mRNAs through deadenylation-dependent or -independent decapping and 5'->3' exonucleolysis or some other mechanism. Sequences or structures necessary for recognition of wild-type mRNAs by NMD will be identified using deletion mutations and gene fusions. The possibility that kinetochore- and/or telomere-related genes may be coordinately regulated by NMD will be investigated through analysis of mRNA from synchronized cell cultures and genetic approaches to identify putative regulatory genes, which may encode mRNA targets of NMD. This project serves as an important starting point to increase understanding of the cellular role of NMD in regulating the expression of wild-type genes and, ultimately, the mechanism for recognition and decay of specific wild-type mRNAs by NMD. In summary, protein molecules are functional components of nearly all the molecular processes in biological systems. Instructions for protein formation are stored in the DNA of genes and are provided as a chemical information intermediate, known as messenger RNA (mRNA), when genes are expressed. To achieve normal growth and development, cells must regulate the expression of genes. Selectively degrading the mRNA of a wild-type gene is an important mechanism for cells to regulate its expression. This research aims to increase understanding of the cellular role for specialized mRNA degradation pathways in regulating wild-type gene expression and the mechanisms involved in recognizing specific wild-type mRNAs for selective degradation.