Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.
Biochemistry. 2020 Apr 21;59(15):1493-1507. doi: 10.1021/acs.biochem.9b01035. Epub 2020 Apr 13.
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 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.
信使 RNA 降解是整体基因表达的一个重要组成部分。在真核生物 mRNA 降解的最后一步中，外切核酸酶 1（Xrn1）利用二价金属离子催化，进行 5'→3' 连续的、水解性的 RNA 分子降解。为了在我们的实验室中开始研究 5'→3' RNA 衰变机制，我们表达了一个 C 端截断的 Xrn1 版本，并使用第二代、时间分辨荧光 RNA 降解测定法探索了它的酶学特性。使用该系统，我们定量地研究了 Xrn1 对 5'-单磷酸化 RNA 底物的偏好性、其 pH 依赖性以及分子保守催化核心中活性位点突变的重要性。此外，我们还研究了 Xrn1 对含有 5'单链区域的 RNA 的偏好性，这些 RNA 存在于分子间发夹结构和 RNA-DNA 杂交双链系统中。这些结果既扩展又巩固了我们对 Xrn1 的理解，Xrn1 是一种重要的酶，其生化特性对许多 RNA 降解和加工途径都有影响。
