BACKGROUND: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions.
RESULTS: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation.
CONCLUSIONS: Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

RNA and protein are interconnected biomolecules that can influence each other\'s life cycles and functions through physical interactions. Abnormal RNA-protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA-protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA-protein complexes manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.