Poster abstracts
Poster number 11 submitted by Robert Connacher
New insights into mRNA regulation by the Drosophila TRIM-NHL protein Brat
Robert P. Connacher (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota), Michael B. OConnor (Dept. Genetics, Cell Biology, and Development, University of Minnesota), Aaron C. Goldstrohm (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota)
Abstract:
Members of the TRIM-NHL family of proteins share a conserved domain architecture and play crucial roles in stem cell biology, fertility, and development. Recently, multiple TRIM-NHLs have been shown to recognize specific RNA motifs and structures via the NHL domain. Functional and genetic analysis shows TRIM-NHLs negatively regulate protein expression of the mRNAs that they bind. However, it is unclear whether RNA-binding is utilized in the multiple developmental processes in which TRIM-NHLs are involved, and how these proteins negatively regulate mRNAs upon binding. These questions were investigated with the Drosophila TRIM-NHL protein Brat. First, RNA-binding defective mutations were introduced into the endogenous brat locus in flies via CRISPR. Alanine substitution of key residues known to abolish RNA-binding in-vitro mimics loss-of-function brat mutations; implying the primary biological function of Brat utilizes RNA-binding. Furthermore, cell culture assays identify three autonomous domains of Brat which exert RNA repression, each utilizing unique means to negatively regulate RNA stability and/or translation. Further analysis of CRISPR mutants and investigation of these autonomous domains will provide a full picture of TRIM-NHL function, at both the transcriptome-wide and molecular levels.
Keywords: Drosophila, CRISPR, development