XUT, a novel class of antisense regulatory ncRNA in yeast

Non-coding (nc)RNAs are key players in numerous biological processes such as gene regulation, chromatin domain formation and genome stability. However, despite recent evidence showing that pervasive transcription is broader than previously thought, only a few antisense long regulatory ncRNAs acting in transcriptional gene silencing in eukaryotes have been described. In S. cerevisiae, the characterized cryptic regulatory ncRNA are controlled by the Xrn1 5'-3' RNA exonuclease, but their generalization remains elusive. Here, using strand-specific RNA-seq, we identify a novel class of 1,658 Xrn1-sensitive Unstable Transcripts (XUTs) in which 66% are antisense to open reading frames (ORF). These transcripts are polyadenylated and RNA Polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in lithium-containing media, suggesting that they might play a role in adaptive responses to physiological stress. Strikingly, RNAPII ChIP-seq analysis of Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over 297 genes with antisense XUT. Those genes show an unusual bias for H3K4me3 mark and require the Set1 histone H3 lysine 4 methyl-transferase for silencing. Furthermore, abolishing H3K4me3 triggers the silencing of other genes with antisense XUT, suggesting that H3K4me3 antagonizes H3K4me2/1 repressive activity. Our results demonstrate that antisense ncRNA-mediated regulation is a general regulatory pathway for gene expression in S. cerevisiae. They support a model where antisense ncRNA-dependent gene regulation is also broadly mediated by trans-acting ncRNA and histone marks.

非编码RNA（non-coding RNAs, ncRNAs）是诸多生物学过程的关键调控因子，参与基因调控、染色质结构域形成以及基因组稳定性维持等核心生理过程。尽管近期研究表明广泛转录的范围远超此前认知，但目前仅在真核生物中报道了少数参与转录基因沉默的反义长调控非编码RNA。在酿酒酵母（S. cerevisiae）中，已鉴定的隐蔽调控非编码RNA均受Xrn1 5'-3'核糖核酸外切酶调控，但其普遍性仍有待阐明。本研究通过链特异性RNA测序技术，在酿酒酵母中鉴定出一类共计1658个全新的Xrn1敏感不稳定转录本（Xrn1-sensitive Unstable Transcripts, XUTs），其中66%的转录本与开放阅读框（open reading frames, ORFs）呈反义方向。此类转录本均为多聚腺苷酸化产物，且为RNA聚合酶II（RNA Polymerase II, RNAPII）依赖型转录本。绝大多数XUTs在含锂培养基中显著富集，提示其可能参与生理胁迫的适应性应答过程。值得注意的是，对Xrn1缺陷菌株的RNA聚合酶II染色质免疫沉淀测序（RNAPII ChIP-seq）分析显示，在297个携带反义XUTs的基因区域内，RNA聚合酶II的结合占有率显著降低。这些基因表现出H3K4me3修饰的异常偏好性，且需要Set1组蛋白H3赖氨酸4甲基转移酶以实现基因沉默。此外，消除H3K4me3修饰会触发其他携带反义XUTs的基因发生沉默，这表明H3K4me3可拮抗H3K4me2与H3K4me1的抑制性修饰活性。本研究结果证实，反义非编码RNA介导的调控是酿酒酵母中基因表达调控的普遍通路。研究结果支持如下模型：反义非编码RNA依赖的基因调控同样可通过反式作用非编码RNA与组蛋白修饰广泛介导。