Dcr1-GFP is functional for 22-23 nt small RNAs production

Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome. Small RNA-Seq analysis in Dcr1-GFP cells of N. castellii.

在缺乏RNA干扰（RNAi，RNA interference）的出芽酵母酿酒酵母（Saccharomyces cerevisiae）中，反义长链非编码RNA（aslncRNAs，antisense long non-coding RNAs）会被核外切体（nuclear exosome）与细胞质外核糖核酸酶Xrn1（exoribonuclease Xrn1）广泛降解。在具备RNAi能力的近缘出芽酵母中，核糖核酸酶III Dicer（ribonuclease III Dicer）是否会对aslncRNAs产生调控作用仍未明确。本研究借助全基因组RNA表达谱分析（genome-wide RNA profiling），针对具备RNAi能力的出芽酵母卡氏纳夫酵母菌（Naumovozyma castellii）展开研究，结果显示aslncRNAs主要受核外切体与Xrn1的靶向调控，而Dicer仅在Xrn1缺陷细胞中影响Xrn1敏感型长链非编码RNA（XUTs，Xrn1-sensitive long non-coding RNAs）的表达水平。dcr1与xrn1突变体表现出协同生长缺陷，这表明在Xrn1缺失的情况下，Dicer会成为细胞生存的关键调控因子。小RNA测序（small RNA sequencing）结果显示，Dicer可将aslncRNAs切割为小RNA，且对asXUTs存在切割偏好性。与之相符的是，Dicer定位于细胞质中。最后，相较于酿酒酵母，卡氏纳夫酵母菌中受核外切体调控的反义转录组出现了扩增，这提示细胞质RNAi的存在强化了细胞核RNA监视系统，以抑制aslncRNAs的表达。本研究的数据为aslncRNAs的代谢调控机制提供了基础性认知，并为探究RNAi在塑造aslncRNAs转录组过程中可能发挥的进化作用开辟了新的研究视角。本数据集包含卡氏纳夫酵母菌Dcr1-GFP细胞的小RNA测序分析。