Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.
Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.
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DOI：
10.1371/journal.pgen.1004288
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发表时间：
2014-04
期刊：
影响因子：
4.5
通讯作者：
Hieter P
中科院分区：
文献类型：
--
作者：
Chan YA;Aristizabal MJ;Lu PY;Luo Z;Hamza A;Kobor MS;Stirling PC;Hieter P
在从细菌到哺乳动物的生物系统中，DNA:RNA杂交形成正在成为基因组不稳定的重要原因。本文采用DNA:RNA免疫沉淀（DRIP）技术，结合微阵列杂交技术，研究了酿酒酵母DNA:RNA杂交易感位点的全基因组分布。这些图谱表明，DNA:RNA杂交体优先聚集在rDNA、Ty1和Ty2转座子、端粒重复区域和开放阅读框（orf）的一个子集上。后者通常转录率高，GC含量高。有趣的是，在与反义转录物相关的基因上也检测到显著的DNA:RNA杂交富集。当RNase H过表达时，反义相关基因的表达也显著改变，而RNase H会降解杂交种中的RNA。最后，我们发现了Sen1解旋酶突变体、RNase H缺失突变体和Hpr1 THO复合物突变体与野生型相比在DRIP谱上的突变特异性差异，表明这些蛋白质在DNA:RNA杂交生物学中的不同作用。我们的DNA:RNA杂交易感位点图谱为了解体内杂交形成区域的特性提供了资源，扩展了我们对杂交缓解酶的认识，并有助于建立反义介导的基因调控模型。本文在第26届酵母遗传学与分子生物学国际会议（2013年8月）上发表。RNA加工因子在人类癌症、遗传性发育障碍和神经退行性综合征中发生突变。RNA加工中的缺陷与突变水平的增加和DNA损伤有关，部分原因是DNA:RNA杂交体的形成。虽然基因组的特定区域可能更容易形成DNA:RNA杂交，但杂交倾向区域的地图是不可用的。在这项研究中，我们描述了正常和突变酿酒酵母细胞中DNA:RNA杂交体的全基因组分布。由此产生的图谱有助于我们对杂交形成位点的一般特性的理解，也有助于我们对杂交缓解酶的了解。有趣的是，在与反义转录相关的基因上检测到显著的DNA:RNA杂交富集。我们发现，RNase H的过表达会显著影响与反义转录物相关的基因的表达，RNase H可以降解杂交种中的RNA。这些发现支持了DNA:RNA杂交体通过反义转录物调控基因表达的作用。
DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. RNA processing factors are mutated in human cancers, inherited developmental disorders and neurodegenerative syndromes. Defects in RNA processing have been associated with increased levels of mutations and DNA damage in part via the formation of DNA:RNA hybrids. Although it is likely that specific regions of the genome are more prone to DNA:RNA hybrid formation, a map of hybrid-prone regions is not available. In this study, we describe the genome-wide distribution of DNA:RNA hybrids in both normal and mutant Saccharomyces cerevisiae cells. The resulting profiles contribute to both our understanding of the general properties of hybrid-forming loci and to our knowledge of hybrid-mitigating enzymes. Interestingly, significant DNA:RNA hybrid enrichment was detected at genes associated with antisense transcription. We show that overexpression of RNase H, which degrades the RNA in hybrids, significantly affects the expression of genes associated with antisense transcripts. These findings support a role for DNA:RNA hybrids in regulation of gene expression by antisense transcripts.
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