Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, F-67084 Strasbourg, France.
Methods. 2013 Sep 15;63(2):135-43. doi: 10.1016/j.ymeth.2013.06.033. Epub 2013 Jul 9.
Ribonucleases play key roles in gene regulation and in the expression of virulence factors in Staphylococcus aureus. Among these enzymes, the double-strand specific endoribonuclease III (RNase III) is a key mediator of mRNA processing and degradation. Recently, we have defined, direct target sites for RNase III processing on a genome-wide scale in S. aureus. Our approach is based on deep sequencing of cDNA libraries obtained from RNAs isolated by in vivo co-immunoprecipitation with wild-type RNase III and two cleavage-defective mutants. The use of such catalytically inactivated enzymes, which still retain their RNA binding capacity, allows the identification of novel RNA substrates of RNase III. In this report, we will summarize the diversity of RNase III functions, discuss the advantages and the limitations of the approach, and how this strategy identifies novel mRNA targets of small non-coding RNAs in S. aureus.
核糖核酸酶在基因调控和金黄色葡萄球菌毒力因子的表达中发挥着关键作用。在这些酶中，双链特异性内切核糖核酸酶 III（RNase III）是 mRNA 加工和降解的关键介质。最近，我们已经在金黄色葡萄球菌中定义了 RNase III 加工的全基因组范围内的直接靶标位点。我们的方法基于使用体内共免疫沉淀与野生型 RNase III 和两种切割缺陷突变体分离的 RNA 进行 cDNA 文库的深度测序。使用这种仍然保留其 RNA 结合能力的催化失活酶可以鉴定 RNase III 的新型 RNA 底物。在本报告中，我们将总结 RNase III 功能的多样性，讨论该方法的优点和局限性，以及该策略如何鉴定金黄色葡萄球菌中小非编码 RNA 的新型 mRNA 靶标。
