83 Oral - Gene Regulation
Friday April 08, 11:15 AM - 11:30 AM

Authors:
Hideyuki Komori ¹; John Bugay ¹; Hua Luo ²; Craig Smibert ^2,3; Howard Lipshitz ²; Cheng-Yu Lee ¹

Affiliations:
1) Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA; 2) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 3) Department of Biochemistry, University of Toronto, Toronto, ON, Canada

Keywords:
h. translational regulation; c. neural stem cells

RNA-binding proteins assemble translational control complexes by recruiting their interactors that exert regulatory effects on target mRNAs. How these multi-protein complexes are assembled in a timely manner to promote dynamic changes in gene regulatory programs that promote proper specification of cell identity during key developmental transitions remains poorly understood. During asymmetric division of type II neuroblasts in fly larval brains, RNA-binding protein Brain tumor (BRAT) segregates into one of the neuroblast progeny in which it expedites the decay of NOTCH target gene transcripts allowing for the onset of differentiation less than 60 minutes after division. The tight correlation between BRAT-mediated mRNA decay and differentiation initiation following asymmetric neuroblast division provides an excellent in vivo paradigm for testing temporospatial control of the assembly of BRAT-containing RNA regulatory complex. We confirmed that NOT1 and PAN3, the scaffolding components of two main RNA deadenylation complexes, co-segregate with BRAT in mitotic neuroblasts and that their segregation is BRAT-dependent, consistent with our previous findings that Brat functions together with these proteins to promote the decay of NOTCH target gene transcripts. We also identified Ubiquitin specific protein 5 (USP5) as a novel BRAT interactor by IP-MS experiment using embryonic extract. We confirmed that USP5 co-segregates with BRAT into neuroblast progeny destined to differentiate, and that asymmetric USP5 segregation depends on BRAT. Furthermore, reducing usp5 function enhanced differentiation defects in brat-hypomorphic brains, and complete loss of usp5 leads to supernumerary neuroblast formation phenocopying brat mutant. By using single-molecule fluorescent RNA in situ hybridization, we demonstrated that neuroblast progeny ectopically accumulate Notch target gene transcripts in brat or usp5-mutant brains, supporting the model that USP5 is a key component of the BRAT RNA decay complex. The scaffolding protein Miranda (MIRA) binds and segregates BRAT in mitotic neuroblasts. Consistent with Miranda inhibiting the RNA-binding ability of BRAT in wild-type neuroblasts, we detected co-localization of NOTCH target mRNAs with mutant BRAT that is defective in MIRA-binding in mitotic neuroblasts. We propose that BRAT assembles an enzymatically inactive and non-RNA-binding pre-complex in mitotic neuroblasts, and the BRAT complex becomes activated in neuroblast progeny by recruiting the enzymatic components of the RNA deadenylation complexes and dissociation from MIRA.