STRUCTURAL DETERMINATION OF THE RRM1 DOMAIN FROM THE HUMAN SPLICING FACTOR RBM6

Cancer is one of the leading causes of death in Brazil and worldwide. In 2020, approximately 620,000 cancer cases and 245,000 cancer-related deaths were recorded. Recent studies have shown that tumor cells are capable of reprogramming canonical alternative splicing, leading to changes in their transcriptome. Among human splicing factors, the RBM (RNA-binding motif) protein family stands out, as it is heavily implicated in tumor development. Within this family, RBM6 is described as a tumor suppressor and is found to be downregulated in certain types of cancer (e.g., breast, lung, lymphoma, sarcoma, and pancreatic cancers). RBM6 consists of two RNA recognition motif (RRM) domains, two zinc finger (ZnF) domains, bipartite nuclear localization signals, an OCRE (OCtamer REpeat) domain, and a glycine-rich C-terminal tail. Despite its functional relevance, RBM6 remains the least studied member of the RBM family, including in terms of structural information, which is essential for the rational development of new anti-cancer therapies.Thus, the present work aims to structurally study the first RRM domain (RRM1) of RBM6 (construct RBM6-RRM1_455-538) using Nuclear Magnetic Resonance (NMR) as a first step toward understanding the molecular mechanisms of RBM6 action. RBM6-RRM1 was expressed in E. coli BL21 (DE3) strains, fused to an N-terminal expression and purification tag. For the production of isotopically enriched samples with ¹⁵N and/or ¹³C, minimal M9 culture medium supplemented with ¹⁵NH₄Cl and/or ¹³C-glucose was used. The construct was purified through nickel affinity chromatography and size-exclusion chromatography. All chromatographic steps were monitored by SDS-PAGE. The highly pure sample was subjected to two- and three-dimensional triple resonance NMR experiments. The 2D [¹H,¹⁵N] HSQC spectrum of RBM6-RRM1 showed well-dispersed, well-resolved, and uniformly intense signals, indicating that the protein was folded in solution. Unambiguous backbone assignments were obtained for 97.7% of the sequence (with the exception of G453) and 82.7% of the side chains. The three-dimensional structure of RBM6-RRM1 has been determined and is undergoing refinement steps. Dynamics experiments (¹⁵N-R₁, ¹⁵N-R₂, ¹⁵N-¹H-heteronuclear NOE and relaxation dispersion) have been acquired, but are yet to be analyzed.