Märtens Birgit, Sharma Kundan, Urlaub Henning, Bläsi Udo
Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, Center of Molecular Biology, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria.
Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen, Germany.
Nucleic Acids Res. 2017 Sep 6;45(15):8957-8967. doi: 10.1093/nar/gkx581.
Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism in Pro- and Eukaryotes. In this study, a collection of 53 mRNAs that co-purified with Sulfolobus solfataricus (Sso) SmAP2 were surveyed for a specific RNA binding motif (RBM). SmAP2 was shown to bind with high affinity to the deduced consensus RNA binding motif (SmAP2-cRBM) in vitro. Residues in SmAP2 interacting with the SmAP2-cRBM were mapped by UV-induced crosslinking in combination with mass-spectrometry, and verified by mutational analyses. The RNA-binding site on SmAP2 includes a modified uracil binding pocket containing a unique threonine (T40) located on the L3 face and a second residue, K25, located in the pore. To study the function of the SmAP2-RBM in vivo, three authentic RBMs were inserted in the 3'UTR of a lacS reporter gene. The presence of the SmAP2-RBM in the reporter-constructs resulted in decreased LacS activity and reduced steady state levels of lacS mRNA. Moreover, the presence of the SmAP2-cRBM in and the replacement of the lacS 3'UTR with that of Sso2194 encompassing a SmAP2-RBM apparently impacted on the stability of the chimeric transcripts. These results are discussed in light of the function(s) of eukaryotic Lsm proteins in RNA turnover.
Sm 蛋白和 Sm 样蛋白代表了一个在进化上保守的家族，在原核生物和真核生物的 RNA 代谢中发挥关键作用。在本研究中，对与嗜热栖热菌（Sso）SmAP2 共纯化的 53 种 mRNA 进行了调查，以寻找特定的 RNA 结合基序（RBM）。结果表明，SmAP2 在体外能与推导的共有 RNA 结合基序（SmAP2-cRBM）高亲和力结合。通过紫外线诱导交联结合质谱法对 SmAP2 中与 SmAP2-cRBM 相互作用的残基进行了定位，并通过突变分析进行了验证。SmAP2 上的 RNA 结合位点包括一个修饰的尿嘧啶结合口袋，其中含有位于 L3 面上的独特苏氨酸（T40）和位于孔中的第二个残基 K25。为了研究 SmAP2-RBM 在体内的功能，将三个真实的 RBM 插入到 lacS 报告基因的 3'UTR 中。报告构建体中 SmAP2-RBM 的存在导致 LacS 活性降低和 lacS mRNA 的稳态水平降低。此外，SmAP2-cRBM 的存在以及用包含 SmAP2-RBM 的 Sso2194 的 3'UTR 替换 lacS 的 3'UTR 显然影响了嵌合转录本的稳定性。根据真核生物 Lsm 蛋白在 RNA 周转中的功能对这些结果进行了讨论。
