Characterization of newly discovered Staufen-mediated RNA decay pathway

Woeller, Collynn

Abstract

? The goal of this research is to identify the components of the newly discovered Staul-mediated mRNA decay (SMD) pathway so that we will gain a better understanding of its physiological significance and role in post-transcriptional gene regulation. Post-transcriptional gene regulation is increasingly becoming recognized as a crucial aspect of cellular homeostasis. Recently, a new pathway of post-transcriptional gene regulation has been discovered in the Maquat laboratory. The double-stranded RNA binding protein Staufenl (Stau) 1 decreases the levels of up to 1% of all transcripts expressed in human HeLa cells. Staul mediates the degradation of these transcripts in a manner that requires the RNA helicase, Upf1. Notably, Upf1 is an essential component of another mRNA degradation pathway, nonsense-mediated mRNA decay (NMD). Apart from a dependence on Staul and Upf1, little is known about the components or molecular dynamics of SMD. Identifying the other components of SMD and the RNA sequences that bind Staul will increase our understanding of the process.
The specific aims are: I. To characterize the complex of proteins that binds the 3' untranslated regions (UTRs) of transcripts targeted for SMD. The complex of proteins that is required for the SMD of several mRNAs will be elucidated and compared. Identifying the proteins involved in SMD will provide important information on the mechanism of SMD. II. To determine the molecular dynamics of Staul-Upf1 complex formation during SMD. Upf1 is a phosphoprotein but the majority of cellular Upf1 is hypophosphorylated. Since Upf1 is activated by phosphorylation during NMD by the protein kinase SMG-1, we will determine if SMG-1 also phosphorylates Upf1 during SMD. Preliminary results suggest Staul is post-translationally modified by arginine methylation. We will determine if Staul is methylated and if methylation affects the ability of Staul to bind Upf1 or otherwise function in SMD. III. To define the RNA sequences/structures that associate with the SMD complex. By generating and analyzing progressive 50-bp deletions and site-directed mutations, and using computational bioinformatics, a consensus SBS will be defined, if there is one. Relevance: Staul has been shown to regulate numerous genes at the mRNA level and has the capacity to be a master regulator of cell growth, division and differentiation. It is important that we elucidate the mechanism whereby Staul and, in particular, SMD regulates gene expression to determine their roles in cellular processes. We believe we are the only group researching this field at the present time. ? ? ? ?