Dynamic imaging and targeted degradation of the RNA‐binding protein hnRNP A2B1 using nanobodies

Abstract Background RNA‐binding proteins (RBP) are a primary component of stress granules, a key indicator of various cell stress responses. Recently, the heterogeneous nuclear ribonucleoprotein hnRNP A2B1 emerged as a primary protein that preferentially interacts with tau oligomers than tau monomers. Here we studied the dynamics of hnRNP A2B1 in cultured cells and primary neurons using a novel nanobody reagent that enables live‐cell imaging. Method We screened a recombinant nanobody library to identify nanobodies (Nbs) that bind to hnRNP A2B1. The Nbs were designed to bind to all isoforms of hnRNP A2B1. We applied protein engineering approaches to improve the binding affinity of the identified Nbs. Live cell imaging was conducted in cultured cell lines and primary mouse hippocampal neurons. We also generated Nb‐E3 ligase fusions for targeted degradation of hnRNP A2B1. Result The Nbs revealed an unexpected localization of hnRNP A2B1 previously not observed using GFP fusions. We observed bright puncta throughout the cytoplasm in addition to typical nucleoplasmic distribution of hnRNP A2B1. The hnRNP A2B1 puncta were co‐localized with exosomal markers. In cell lines, the nucleoplasmic hnRNP A2B1 was dramatically reduced during mitosis, while the cytoplasmic puncta did not change, suggesting cell cycle‐dependent nucleocytoplasmic translocation. The Nb‐E3 ligase fusion constructs caused a significant reduction in hnRNP A2B1, lowering the level by 60%. Conclusion These novel tools will greatly aid the study of endogenous hnRNP A2B1 dynamics and function. Isoform‐dependent localization of hnRNP A2B1 and whether it is involved in protein loading to exosomes remains to be studied.