What does it take to make a B-body?
Disciplines
Cell and Developmental Biology | Genetics and Genomics
Abstract (300 words maximum)
Nuclear domains are distinct compartments within the nucleus that selectively concentrate specific proteins, yet their regulation remains largely unclear. One such domain, B-body, forms in Drosophila flight muscles and contains the RNA-binding protein Bruno (Bru). Previous work in our lab identified the long non-coding RNA Hsr-omega as a scaffolding molecule for B-body. Here, we investigate which regions of Hsr-omega and Bru are essential for B-body formation. Using bioinformatics, we selected several Hsr-omega regions: the conserved 5’ region (Hsr5) found in all isoforms, the core repetitive region (RR) present in long isoforms, and three extended regions (RF1, RF2, and RF3) unique to the longest isoform, Hsr-omega-RF. We cloned and expressed these regions in flight muscles, then used immunofluorescence and in situ fluorescence hybridization to assess their co-localization with Bru. Additionally, we expressed Bru mutants to identify the protein regions required for Hsr-omega interaction. None of the truncated Hsr-omega constructs were able to interact with Bru to form B-body-like structures. However, the ability of Bru to bind Hsr-omega in B-bodies was mapped to its RNA-Recognition Motif 2 (RRM2). These findings suggest that protein recruitment into nuclear domains depends on RNA-binding specificity, while RNA's ability to organize nuclear domains is length-dependent.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Dr. Anton Bryantsev
What does it take to make a B-body?
Nuclear domains are distinct compartments within the nucleus that selectively concentrate specific proteins, yet their regulation remains largely unclear. One such domain, B-body, forms in Drosophila flight muscles and contains the RNA-binding protein Bruno (Bru). Previous work in our lab identified the long non-coding RNA Hsr-omega as a scaffolding molecule for B-body. Here, we investigate which regions of Hsr-omega and Bru are essential for B-body formation. Using bioinformatics, we selected several Hsr-omega regions: the conserved 5’ region (Hsr5) found in all isoforms, the core repetitive region (RR) present in long isoforms, and three extended regions (RF1, RF2, and RF3) unique to the longest isoform, Hsr-omega-RF. We cloned and expressed these regions in flight muscles, then used immunofluorescence and in situ fluorescence hybridization to assess their co-localization with Bru. Additionally, we expressed Bru mutants to identify the protein regions required for Hsr-omega interaction. None of the truncated Hsr-omega constructs were able to interact with Bru to form B-body-like structures. However, the ability of Bru to bind Hsr-omega in B-bodies was mapped to its RNA-Recognition Motif 2 (RRM2). These findings suggest that protein recruitment into nuclear domains depends on RNA-binding specificity, while RNA's ability to organize nuclear domains is length-dependent.