Intronic RNA secondary structural information captured for the human MYC pre-mRNA.

IF 4 Q1 GENETICS & HEREDITY NAR Genomics and Bioinformatics Pub Date : 2024-10-24 eCollection Date: 2024-09-01 DOI:10.1093/nargab/lqae143
Taylor O Eich, Collin A O'Leary, Walter N Moss
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Abstract

To address the lack of intronic reads in secondary structure probing data for the human MYC pre-mRNA, we developed a method that combines spliceosomal inhibition with RNA probing and sequencing. Here, the SIRP-seq method was applied to study the secondary structure of human MYC RNAs by chemically probing HeLa cells with dimethyl sulfate in the presence of the small molecule spliceosome inhibitor pladienolide B. Pladienolide B binds to the SF3B complex of the spliceosome to inhibit intron removal during splicing, resulting in retained intronic sequences. This method was used to increase the read coverage over intronic regions of MYC. The purpose for increasing coverage across introns was to generate complete reactivity profiles for intronic sequences via the DMS-MaPseq approach. Notably, depth was sufficient for analysis by the program DRACO, which was able to deduce distinct reactivity profiles and predict multiple secondary structural conformations as well as their suggested stoichiometric abundances. The results presented here provide a new method for intronic RNA secondary structural analyses, as well as specific structural insights relevant to MYC RNA splicing regulation and therapeutic targeting.

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捕捉到的人类 MYC 前核糖核酸内部二级结构信息。
为了解决人类 MYC pre-mRNA 二级结构探测数据中缺乏内含子读数的问题,我们开发了一种将剪接体抑制与 RNA 探测和测序相结合的方法。Pladienolide B 与剪接体的 SF3B 复合物结合,抑制剪接过程中的内含子去除,从而保留了内含子序列。这种方法用于提高 MYC 内含子区域的读数覆盖率。提高内含子覆盖率的目的是通过 DMS-MaPseq 方法生成内含子序列的完整反应谱。值得注意的是,DRACO 程序的深度足以进行分析,该程序能够推导出不同的反应性曲线,并预测多种二级结构构象及其建议的化学丰度。本文介绍的结果为内含子 RNA 二级结构分析提供了一种新方法,也为 MYC RNA 剪接调控和靶向治疗提供了特定的结构见解。
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来源期刊
CiteScore
8.00
自引率
2.20%
发文量
95
审稿时长
15 weeks
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