Events

Structural and Functional Studies of T-Cell Intracellular Antigen-1 (TIA1)

Presented By: Yizhuo Yang

Abstract:
T-Cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. It contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this thesis aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2 but also weakly at RRM3, and Zn2+ binds primarily to RRM3. Binding of Zn²⁺ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn²⁺ caused the TIA1:DNA complexes to collapse, indicating that Zn²⁺ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3. Furthermore, I investigated the interdomain interactions between RRM2 and RRM3 by successfully preparing segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn²⁺ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn²⁺ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.