Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096, Haifa, Israel.
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT, 06536, USA.
Hum Genomics. 2018 Jun 27;12(1):32. doi: 10.1186/s40246-018-0164-4.
Naturally occurring stress-induced transcriptional readthrough is a recently discovered phenomenon, in which stress conditions lead to dramatic induction of long transcripts as a result of transcription termination failure. In 2015, we reported the induction of such downstream of gene (DoG) containing transcripts upon osmotic stress in human cells, while others observed similar transcripts in virus-infected and cancer cells. Using the rigorous methodology Cap-Seq, we demonstrated that DoGs result from transcriptional readthrough, not de novo initiation. More recently, we presented a genome-wide comparison of NIH3T3 mouse cells subjected to osmotic, heat, and oxidative stress and concluded that massive induction of transcriptional readthrough is a hallmark of the mammalian stress response. In their recent letter, Huang and Liu in contrast claim that DoG transcripts result from novel transcription initiation near the ends of genes. Their conclusions rest on analyses of a publicly available transcription start site (TSS-Seq) dataset from unstressed NIH3T3 cells. Here, we present evidence that this dataset identifies not only true transcription start sites, TSSs, but also 5'-ends of numerous snoRNAs, which are generally processed from introns in mammalian cells. We show that failure to recognize these erroneous assignments in the TSS-Seq dataset, as well as ignoring published Cap-Seq data on TSS mapping during osmotic stress, have led to misinterpretation by Huang and Liu. We conclude that, contrary to the claims made by Huang and Liu, TSS-Seq reads near gene ends cannot explain the existence of DoGs, nor their stress-mediated induction. Rather it is, as we originally demonstrated, transcriptional readthrough that leads to the formation of DoGs.
自然发生的应激诱导转录通读是最近发现的一种现象，即在应激条件下，由于转录终止失败，导致长转录本的显著诱导。2015 年，我们报道了在人类细胞受到渗透胁迫时，这种基因下游（DoG）包含的转录本的诱导，而其他人在病毒感染和癌细胞中也观察到了类似的转录本。使用严格的 Cap-Seq 方法，我们证明了 DoGs 是由转录通读而不是从头起始产生的。最近，我们对 NIH3T3 小鼠细胞进行了全基因组比较，这些细胞受到渗透、热和氧化应激，得出结论：大量诱导转录通读是哺乳动物应激反应的一个标志。在他们最近的信中，Huang 和 Liu 相反地声称，DoG 转录本是由基因末端附近的新转录起始产生的。他们的结论基于对未受应激的 NIH3T3 细胞的公共转录起始位点（TSS-Seq）数据集的分析。在这里，我们提供的证据表明，该数据集不仅可以识别真正的转录起始位点（TSS），还可以识别大量 snoRNAs 的 5'端，这些 snoRNAs 通常在哺乳动物细胞中从内含子中加工而来。我们表明，在 TSS-Seq 数据集中未能识别这些错误的分配，以及在渗透胁迫期间忽略关于 TSS 映射的已发表的 Cap-Seq 数据，导致了 Huang 和 Liu 的错误解释。我们的结论是，与 Huang 和 Liu 的说法相反，基因末端附近的 TSS-Seq 读段不能解释 DoGs 的存在，也不能解释它们的应激介导诱导。相反，正如我们最初所证明的，是转录通读导致了 DoGs 的形成。
