Meszaros, A. | bioRxiv Science

bioRxiv2025

Expression and Purification of Full-Length hnRNPA2B1 for Biophysical Characterization of Liquid-Liquid Phase Separation

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is a multifunctional RNA-binding protein involved in RNA maturation and mRNA transport. It has recently been shown to undergo liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles. Moreover, dysregulation of LLPS is associated with the formation of pathogenic protein aggregates, in which hnRNPA2B1 is frequently found. Despite its biological and pathological relevance, studies on the full-length protein remain limited due to its intrinsically disordered, low-complexity domain, which renders hnRNPA2B1 highly aggregation-prone and difficult to purify. In this study, we report the successful expression and purification of full-length hnRNPA2B1 with high purity and minimal nucleic acid contamination. By optimizing buffer conditions, specifically ionic strength and pH, we maintain the protein in solution following cleavage of its solubility tag. Preliminary in vitro characterization under near-physiological conditions reveals that purified hnRNPA2B1 undergoes LLPS, forming dynamic liquid-like droplets that grow and mature into amorphous aggregates. Our approach provides a robust method for purifying hnRNPA2B1 suitable for LLPS and aggregation studies. This strategy may also be useful to purify other aggregation-prone, intrinsically disordered proteins.

biochemistry↗

bioRxiv2023

Menin regulates androgen receptor- and MLL-driven condensation, upsetting regulation of cellular AR-driven transcription.

Menin is a protein that is regulated via protein-protein interactions by different binding partners, such as mixed lineage leukemia protein (MLL) and androgen receptor (AR). We observed that menin-AR and menin-MLL interactions are regulated by concentration-dependent dimerization of menin, and its interaction with cancer-related AR. As a result of its oligomerization-dependent interaction with both AR and MLL, menin is recruited into AR-RNA and MLL-RNA condensates formed by liquid-liquid phase separation (LLPS), with different outcomes under AR-overexpression or MLL-overexpression conditions representing different cancer types. At high concentrations promoting menin dimerization, it inhibits MLL-RNA LLPS, while making AR-RNA condensates less dynamic, i.e., more gel-like. Regions of AR show both negative/positive cooperativity in menin binding. AR contains a specific menin-binding region (MBR) in its intrinsically disordered N-terminal domain (NTD), menin binding of which is inhibited by the adjacent DNA-binding domain (DBD), but facilitated by a hinge region located between its DBD and ligand-binding domain (LBD) as well as by N terminus of AR. Interestingly, the hinge region reduces the propensity of full-length AR to undergo LLPS in the presence of RNA, which is facilitated by an alternative hinge region present in the tumor-specific AR isoform, AR-v7. As both menin and MLL are recruited into AR-driven, functional cellular condensates aggravated in the case of AR-v7, we posit that the menin-AR-MLL system represents a fine-tuned condensate module of transcription regulation that is balanced toward the tumor-suppressor activity of menin. Our results suggest that this balance can be upset by prevalent oncogenic events, such as menin upregulation and/or AR-v7 overexpression, in cancer.

biochemistry↗