Biochemical Characterization of Yeast Xrn1.

Conner J Langeberg, William R W Welch, John V McGuire, Alison Ashby, Alexander D Jackson, Erich G Chapman

Biochemistry 2020 Apr 21

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Messenger RNA degradation is an important component of overall gene expression. During the final step of eukaryotic mRNA degradation, exoribonuclease 1 (Xrn1) carries out 5' → 3' processive, hydrolytic degradation of RNA molecules using divalent metal ion catalysis. To initiate studies of the 5' → 3' RNA decay machinery in our lab, we expressed a C-terminally truncated version of Saccharomyces cerevisiae Xrn1 and explored its enzymology using a second-generation, time-resolved fluorescence RNA degradation assay. Using this system, we quantitatively explored Xrn1's preference for 5'-monophosphorylated RNA substrates, its pH dependence, and the importance of active site mutations in the molecule's conserved catalytic core. Furthermore, we explore Xrn1's preference for RNAs containing a 5' single-stranded region both in an intermolecular hairpin structure and in an RNA-DNA hybrid duplex system. These results both expand and solidify our understanding of Xrn1, a centrally important enzyme whose biochemical properties have implications in numerous RNA degradation and processing pathways.

Citation

Conner J Langeberg, William R W Welch, John V McGuire, Alison Ashby, Alexander D Jackson, Erich G Chapman. Biochemical Characterization of Yeast Xrn1. Biochemistry. 2020 Apr 21;59(15):1493-1507