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首页> 外文期刊>Journal of Molecular Biology >The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast
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The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

机译：S9.6抗体对双链RNA的亲和力会影响裂变酵母中R环的准确映射

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R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions. (C) 2017 Elsevier Ltd. All rights reserved.
机译：R-LOOPS，由稳定DNA的形成产生：RNA杂种可以威胁到基因组完整性，并充当基因表达和染色质图案的生理调节剂。为了在裂变酵母中表征R圈，我们使用S9.6抗体的DRIPC-SEQ方法来序列R-LOOPS的RNA链，并在接近核苷酸分辨率下获得链特异性的R环图。令人惊讶的是，初步DRIPC-SEQ实验主要鉴定出抗性的耐呼曲面，但外出敏感的RNA，其映射到DNA链和类似的RNA杂交种（DSRNA），表明DSRNA在裂变酵母中广泛形成DSRNA。我们在体外证实了S9.6可以免疫沉淀的DSRNA，并提供证据表明DSRNA可以干扰其与R圈的结合。 DSRNA通过RNase III治疗在DRIPC-SEQ之前进行消除，允许基因组和基因特异性鉴定真正的R环鉴定，其在体内对RNase H级别进行响应，并显示与R环形成相关的经典特征。我们还发现，大多数通过体内操纵RNase H水平改变的数量的转录物未形成可检测的R环，表明R环水平的长时间操纵可以间接改变转录组。我们讨论了我们在设计实验策略设计中对探测R环功能的影响。 （c）2017 Elsevier Ltd.保留所有权利。

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