The objective of this project is to provide a detailed view of the interactions between the human Staufen protein, which contains multiple double-stranded RNA binding domains, and its Staufen-mediated decay (SMD) RNA target. The Maquat lab has discovered that Staufen regulates the abundance of several mRNAs by binding to their 3'untranslated regions at a site called the Staufen binding site (SBS). mRNA decay is elicited in a way that depends on the nonsense-mediated mRNA decay (NMD) factor Upfl. The best characterized SBS is within the 3'UTR of the ARF1 mRNA, where this region can replace the 3'end of a reporter mRNA and causes its degradation in vivo in a Staufen-and Upf1-dependent manner. There are hundreds of targets for Staufen binding identified by: 1) microarray experiments based on RNAs associated with immunopurified Staufen;2) RT-PCR analysis of RNA co-immunopurifed with Staufen;and 3) recent unpublished data (CLIP) describing the exact RNA sequences that are cross-linked with Immunopurified Staufen. While mutational studies of the ARF1 SBS have been performed to determine some of the secondary structure requirements for Staufen binding, no sequence commonality between the various Staufen targets has been found, suggesting that recognition of the RNA is based on its secondary or tertiary structure.
The specific aims are as follows: 1) determine the structure of ARF1 SBS RNA by using selective 2'hydroxyl acylation analyzed by primer extension (SHAPE) and RNase probing experiments, where the data can be incorporated into the RNAstructure program (developed at the University of Rochester) to deduce an experimentally supported secondary structure, and then combining this data with small angle X- ray scattering (SAXS) data to determine the overall three-dimensional shape, and 2) determine the regions of interaction between Staufen and ARF1 SBS RNA by directed hydroxyl radical footprinting and RNase footprlnting assays as well as obtaining an atomic description of the Staufen-ARF1 SBS RNA complex using X-ray crystallography. This proposal is in line with the mission of the Division of Cell Biology and Biophysics of NIGMS in that it is aimed at understanding the structures of both protein and RNA and the complex of the two to elucidate a common theme of RNA recognition that is responsible for RNA metabolism and differentiation in human cells. Relevance: Proper functioning of a human cell requires controlling mRNA abundance during growth and differentiation. In order to further understand SMD, the structure of the canonical ARF1 SBS RNA target must be understood, as well as its specific interaction with Staufen
|Gleghorn, Michael L; Gong, Chenguang; Kielkopf, Clara L et al. (2013) Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay. Nat Struct Mol Biol 20:515-24|
|Park, Eonyoung; Gleghorn, Michael L; Maquat, Lynne E (2013) Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity. Proc Natl Acad Sci U S A 110:405-12|