STRUCTURAL CHARACTERIZATION OF THE RRM2 DOMAIN OF THE SPLICING REGULATORY PROTEIN RBM6

RBM6, RNA-Binding Motif 6, is a nuclear protein involved in various RNA metabolism processes such as alternative splicing, mRNA stabilization, and transcriptional regulation. Additionally, RBM6 is known to participate in the regulation of apoptosis in healthy cells. Interestingly, the expression of this protein is reduced in several cancer types (e.g., lung cancer). Moreover, RBM6 has been implicated as a potential biomarker for early diagnosis in pancreatic cancer. Given its association with this complex disease, it is necessary to better understand the structure-function relationship of RBM6 within the cell to pave the way for the future development of new and effective anticancer therapies.RBM6 consists of two N-terminal RRM (RNA Recognition Motif) domains (RRM1 and RRM2), two ZnF (Zinc Finger) domains, one OCRE (Octamer Repeat of aromatic residues) domain, and a glycine-rich C-terminal tail. This work focuses on the RRM2 domain of RBM6, aiming to obtain structural information and insights into its interaction with nucleic acids. An RBM6 RRM2 construct comprising residues 652 to 743, was fused to an N-terminal His6TEV expression/purification tag (6 histidines plus a TEV protease cleavage site). The construct was expressed solubly in Escherichia coli BL21(DE3) at 18 °C for 18 hours and purified using a combination of chromatographic techniques. The first step was nickel affinity chromatography followed by cleavage of the N-terminal tag using His6-TEV protease. The cleaved N-terminal tag and the His6-TEV protease were then removed by a second nickel affinity purification step, allowing collection of the RBM6 RRM2 construct in the wash fraction. Finally, the construct was subjected to size-exclusion chromatography. Two-dimensional [¹H, ¹⁵N] HSQC Nuclear Magnetic Resonance (NMR) spectrum of ¹⁵N-labeled RBM6 RRM2 was acquired. It showed well-dispersed resonances with modulated intensities, indicating that the recombinant proteins were well folded in solution. Additionally, there is evidence suggesting internal dynamic events on the intermediate NMR timescale, which may indicate conformational exchange processes. Currently, we are collecting additional NMR data required for resonance assignment, a crucial step in the NMR structural determination process and necessary for analyzing the domain’s dynamics. These are the first steps toward a deeper understanding of the RBM6 mechanism of action.