Biochemical characterization of the human mitochondria helicase Suv3

Autor: Wei-Hsuan Tang, 唐偉軒
Rok vydání: 2019
Druh dokumentu: 學位論文 ; thesis
Popis: 107
Mitochondria are indispensable organelles in all eukaryotes as they provide cellular energy in the form of ATP. Defects in mitochondrial functions thus lead to various mitochondria disorder and human diseases. To maintain the normal function of mitochondria, RNA turnover plays an important part in the regulation of mitochondrial gene expression and the surveillance of abnormal RNA molecules. In human mitochondria, dimeric helicase Suv3 and trimeric polynucleotide phosphorylase (PNPase) form a pentameic RNA exosome for unwinding and degrading structured RNA from 3'' to 5'' ends. Although the structure of monomeric Suv3 was reported, it remains unclear how Suv3 is assembled into a homodimer to interact with PNPase in the mitochondrial RNA exosome for corporative RNA unwinding and degradation. To understand how Suv3 unwinds a RNA duplex, we expressed human Suv3 (residues 44-786) and a C-terminal tail-truncated Suv3, named Suv3ΔC (residues 44-722) in E. coli, both without the N-terminal mitochondrial localization sequence (MLS). The purified recombinant Suv3 and Suv3ΔC formed dimers and monomers, respectively, suggesting that the C-terminal tail of Suv3 mediates protein dimerization. We found that Suv3 had a 10-fold greater RNA-binding affinity compared with Suv3ΔC at pH7.4. Suv3 bound single-stranded RNA with high affinities in the presence or absence of ATP and ADP, while Suv3ΔC bound RNA with a high affinity only in the presence of ATP. Suv3 had a slightly higher ATP-dependent helicase activity than Suv3ΔC in unwinding a duplex RNA with a 3''-overhang. Dimeric conformation of Suv3 is important not only for promoting the ATP-dependent RNA helicase activity but also for interaction with PNPase. Taken together these results suggest that RNA is continuously bound with Suv3 after ATP hydrolysis and RNA unwinding, and the unwound single-stranded RNA is degraded processively by the Suv3-interacting PNPase. We thus conclude that dimeric Suv3 and trimeric PNPase form a robust machinery of mitochondrial RNA exosome for efficient processive RNA unwinding and degradation.
Databáze: Networked Digital Library of Theses & Dissertations
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