INITIAL STRUCTURAL CHARACTERIZATION OF THE TUMORIGENIC SPLICING REGULATOR RALY

RALY (RNA-binding protein Associated with Lethal Yellow mutation) is a member of the heterogeneous ribonucleoprotein (hnRNP) family, involved in various RNA metabolism processes such as alternative splicing, mRNA stabilization, and transcriptional and translational control. RALY interacts with the spliceosome and regulates the splicing of several key transcriptional targets, including TP53 (tumor suppressor), NUMB (development), FAS (apoptosis), CDK5R1 (cell cycle), and MTA1 (metastasis). Overexpression of RALY has been observed in multiple cancer types, and there is a significant correlation between RALY overexpression and poor prognosis in ovarian, lung, bladder, brain, breast, and liver cancers. Thus, understanding the structure-function relationship of RALY is crucial for developing novel and effective anti-cancer therapies. Here, we expressed and purified two constructs of RALY to determine their structure, characterize their internal dynamics, and investigate their RNA interaction specificity. The constructs included one containing the isolated N-terminal RNA recognition motif (RRM) domain (residues 1-97) and another containing the RRM domain plus an intrinsically disordered region (residues 1-141). Both constructs were cloned into the RP1B vector, which fuses the protein of interest with an N-terminal Thio₆His₆TEV expression/purification tag. The RALY constructs were expressed solubly in Escherichia coli BL21(DE3) at 18 °C for 18 hours. His₆-RALY_1-97 and His₆-RALY_1-141 were purified via nickel-affinity chromatography, followed by cleavage of the N-terminal tag using His₆-TEV protease. The cleaved His₆-tag and His₆-TEV protease were removed by a second nickel-affinity purification step, allowing the RALY constructs to flow through the column. Finally, the RALY constructs were further purified by size-exclusion chromatography. We acquired [¹H,¹⁵N] HSQC spectra of both ¹⁵N-RALY_1-97 and ¹⁵N-RALY_1-141. The spectra exhibited well-dispersed resonances with homogeneous intensities, indicating that the recombinant proteins are well-folded in solution. We are currently collecting the necessary NMR data for resonance assignment to further characterize the structure and dynamics of the RALY constructs, aiming to elucidate their mechanisms of splicing regulation.