510V Poster - 06. Regulation of gene expression
Wednesday April 06, 4:00 PM - 7:00 PM

Authors:
Robert Connacher ¹; Yichao Hu ^2,3; Richard Roden ¹; Xiaohang Yang ³; Howard Lipshitz ²; Michael O'Connor ¹; Aaron Goldstrohm ¹

Affiliations:
1) University of Minnesota, Minneapolis, MN; 2) University of Toronto, Toronto, ON, Canada; 3) Zhejiang University, Hangzhou, China

Keywords:
u. RNA binding proteins; h. translational regulation

TRIM-NHL proteins share a conserved domain architecture and play crucial roles in stem cell biology, fertility, and development. Recently, multiple TRIM-NHL proteins were shown to recognize specific RNA motifs and structures via their NHL domain. TRIM-NHLs negatively regulate their bound mRNAs. In light of this observation, we sought to determine the biological role of their RNA-binding activity. Additionally, we investigated the mechanism by which TRIM-NHLs repress mRNAs.

We focused on the Drosophila TRIM-NHL protein Brain Tumor (BRAT), which controls development and stem cell fate. First, RNA-binding defective mutations were introduced into the endogenous brat locus via CRISPR/Cas9 genome engineering. Our phenotypic analysis demonstrated that the key residues necessary for RNA-binding in vitro are essential for survival and larval neurogenesis. RNA-binding defective mutations phenocopy the lethality observed in loss-of-function brat mutations. Additionally, these mutations produce ectopic neuroblasts and brain tumors similar to classical brat mutants. These results demonstrate the essential function of BRAT derives from its ability to bind and regulate mRNAs.

We further used cell-based reporter assays to elucidate the molecular mechanism by which BRAT controls protein expression. We find that BRAT-mediated repression depends on the 3’ poly-adenosine tail of target mRNAs and components of the CCR4-NOT deadenylase complex. We also identified the domains of BRAT that are necessary to repress target mRNAs. Together, our data support a mechanism wherein BRAT recruits deadenylases to reduce translation and promote decay of target mRNAs.

Collectively, our findings provide crucial insights into the molecular mechanism and function of BRAT in vivo.