Cortázar Michael A, Fong Nova, Bentley David L
Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, USA.
Bio Protoc. 2023 Sep 20;13(18):e4828. doi: 10.21769/BioProtoc.4828.
In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5'-monophosphorylated (5'-PO) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5'-PO ends can be a target of further processing or be subjected to 5'-3' exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5'-PO ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5'-PO RNA ends with the 3'-hydroxyl group of a 5' adaptor (5'-PO Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5'-PO groups precisely. This protocol successfully detects the 5'monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5'-3' exonuclease Xrn2, 5'-PO Bru-Seq maps the 5' splice sites of debranched introns and mRNA and tRNA 3' end processing sites cleaved by CPSF73 and RNaseZ, respectively. Key features • Metabolic labeling for brief periods with bromouridine focuses the analysis of 5'-PO RNA ends on the population of nascent transcripts that are actively transcribed. • Detects 5'-PO RNA ends on nascent transcripts produced by all RNA polymerases. • Detects 5'-PO RNA ends at single nucleotide resolution.
在真核细胞中，RNA生物合成通常需要在RNA聚合酶合成新生转录本的过程中对其进行加工。这些加工事件包括内切核酸酶切割、外切核酸酶修剪以及对正在生长的新生转录本进行剪接。在rRNA、tRNA和mRNA的成熟过程中会发生内切核酸酶切割事件，从而在正在生长的新生转录本上产生一个暴露的5'-单磷酸化（5'-PO）末端。这些5'-PO末端可以成为进一步加工的靶点，或者受到5'-3'外切核酸酶消化，这可能导致转录终止。在这里，我们描述了如何鉴定新生RNA代谢中间体的5'-PO末端。我们通过用溴尿苷进行代谢标记来捕获这些中间体，随后进行免疫沉淀，并使用RNA连接酶I将5'-PO RNA末端与5'接头的3'-羟基进行特异性连接（5'-PO Bru-Seq）。这些连接事件通过高通量测序定位到单核苷酸分辨率，从而精确鉴定5'-PO基团的位置。该方案成功地检测到了成熟核糖体RNA、转运RNA和微小RNA产生过程中RNA加工中间体的5'-单磷酸化末端。当与核5'-3'外切核酸酶Xrn2的抑制相结合时，5'-PO Bru-Seq可以分别绘制出被去分支内含子的5'剪接位点以及由CPSF73和RNaseZ切割的mRNA和tRNA 3'末端加工位点。关键特性 • 用溴尿苷进行短时间代谢标记，将对5'-PO RNA末端的分析聚焦于活跃转录的新生转录本群体。 • 检测所有RNA聚合酶产生的新生转录本上的5'-PO RNA末端。 • 以单核苷酸分辨率检测5'-PO RNA末端。
