Bruno and B-bodies: Unraveling Nuclear Domain Formation
Disciplines
Cell Biology | Integrative Biology
Abstract (300 words maximum)
Nuclear domains are membraneless organelles found in the nuclei of eukaryotic cells. The B-body is a nuclear domain that accumulates the protein Bruno (Bru) and exists in the developing flight muscles in Drosophila. We used the B-body and Bru as a model to study formation of nuclear domains. Specifically, we searched for the amino acid sequences that make Bru accumulate in B-bodies. We used genetic engineering to create various Bru mutants, which were subsequently tested in flies. Nuclear distribution of mutants was analyzed by cryosectioning, immunostaining, and fluorescence microscopy. Three RRM domains determine Bru binding to RNA. We determined that all RRMs must be intact for Bru to interact with the RNA scaffold inside the B-body. Therefore, RNA determines the specificity of protein accumulation in B-bodies. We also tested the role of two unstructured protein regions, called IDR1 and IDR2. Normally, IDRs assist with liquid-liquid phase separation and formation of protein inclusions. Indeed, IDR2 was important for forming nuclear inclusions, however IDR1 was not. Using a series of truncation mutants, we identified a putative regulatory region within IDR1 that controls the ability of Bru to form inclusions. We believe this region is controlled by phosphorylation. Our results help to better understand the formation and regulation of nuclear domains.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Anton Bryantsev
Bruno and B-bodies: Unraveling Nuclear Domain Formation
Nuclear domains are membraneless organelles found in the nuclei of eukaryotic cells. The B-body is a nuclear domain that accumulates the protein Bruno (Bru) and exists in the developing flight muscles in Drosophila. We used the B-body and Bru as a model to study formation of nuclear domains. Specifically, we searched for the amino acid sequences that make Bru accumulate in B-bodies. We used genetic engineering to create various Bru mutants, which were subsequently tested in flies. Nuclear distribution of mutants was analyzed by cryosectioning, immunostaining, and fluorescence microscopy. Three RRM domains determine Bru binding to RNA. We determined that all RRMs must be intact for Bru to interact with the RNA scaffold inside the B-body. Therefore, RNA determines the specificity of protein accumulation in B-bodies. We also tested the role of two unstructured protein regions, called IDR1 and IDR2. Normally, IDRs assist with liquid-liquid phase separation and formation of protein inclusions. Indeed, IDR2 was important for forming nuclear inclusions, however IDR1 was not. Using a series of truncation mutants, we identified a putative regulatory region within IDR1 that controls the ability of Bru to form inclusions. We believe this region is controlled by phosphorylation. Our results help to better understand the formation and regulation of nuclear domains.