MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing
MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing
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DOI：
10.1016/j.isci.2020.101318
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发表时间：
2020-07-24
期刊：
影响因子：
5.8
通讯作者：
Singer, Robert H.
中科院分区：
文献类型：
--
作者：
Biswas, Jeetayu;Rahman, Reazur;Singer, Robert H.
紫外交联和免疫沉淀（CLIP）和RNA编辑（TRIBE）都可以识别RNA结合蛋白的靶标。为了评估CLIP和TRIBE的假阳性，用MS 2茎环标记内源性β-肌动蛋白mRNA，使其成为MS 2衣壳蛋白（MCP）的唯一真正靶mRNA。CLIP和TRIBE检测到β-肌动蛋白，尽管有假阳性。假阳性CLIP信号归因于非特异性抗体相互作用。相比之下，假定的假阳性TRIBE靶标是在空间上接近0-肌动蛋白基因的基因。MCP-ADAR编辑了附近的新生转录本，与Hi-C中观察到的染色体间接触一致。新生接触的鉴定意味着RNA调节蛋白（例如，剪接因子）与多种新生转录物相关，形成转录后活性结构域。用整合的诱导型MS 2报告基因重复这些结果表明，MS 2-TRIBE可以应用于广泛的细胞和转录本，以研究空间组织和核RNA调控。
Both UV-cross-linking and immunoprecipitation (CLIP) and RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives of CLIP and TRIBE, endogenous beta-actin mRNA was tagged with MS2 stem loops, making it the only bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE detected beta-actin, albeit with false-positives. False-positive CLIP signals were attrib-uted to nonspecific antibody interactions. In contrast, putative false-positive TRIBE targets were genes spatially proximal to the 0-actin gene. MCP-ADAR edited nearby nascent transcripts consistent with interchromosomal contacts observed in Hi-C. The identification of nascent contacts implies RNA regulatory proteins (e.g., splicing factors) associated with multiple nascent transcripts, forming domains of post-transcriptional activity. Repeating these results with an integrated inducible MS2 reporter indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts to study spatial organization and nuclear RNA regulation.