Circularized RNA sequencing of wild-type and Mrpp3 knock out mouse heart mitochondria

Here we find that circularization of RNA prior to deep sequencing enables the discovery and characterization of unprocessed mitochondrial RNAs. Using this approach we identify the most stable processing intermediates and the presence of intermediate processing products that are partially degraded and polyadenylated. Analysis of libraries constructed using RNA from mice lacking the nuclease subunit of the mitochondrial RNase P reveals the identities of stalled processing intermediates, their order of cleavage, and confirms the importance of RNase P (MRPP3) in generating mature mitochondrial RNAs. Using RNA circularization prior to library preparation should provide a generally useful approach to studying RNA processing in many different biological systems. RNA from purified mouse mitochondria from wild-type and Mrpp3 knock out mouse hearts was circularized and the resulting libraries were sequenced using an Illumina MiSeq.本研究发现，在深度测序（deep sequencing）前对RNA进行环化处理，可实现未加工线粒体RNA的发现与特征表征。利用该方法，我们鉴定出了最稳定的RNA加工中间产物，以及发生部分降解并带有多聚腺苷酸化（polyadenylated）修饰的中间加工产物。对缺失线粒体核糖核酸酶P（mitochondrial RNase P）核酸酶亚基的小鼠来源RNA所构建的测序文库进行分析，我们明确了停滞型加工中间产物的具体种类、其切割顺序，并证实了核糖核酸酶P（RNase P, MRPP3）在生成成熟线粒体RNA过程中的关键作用。在文库制备前对RNA进行环化处理，有望成为一种普适性的实用方法，用于诸多不同生物系统中的RNA加工研究。本研究中，我们对从野生型及Mrpp3基因敲除小鼠心脏中纯化得到的线粒体RNA进行了环化处理，构建得到的测序文库采用因美纳（Illumina）MiSeq测序平台完成测序。