Xrn1p Acts at Multiple Steps in the Budding-Yeast RNAi Pathway to Enhance the Efficiency of Silencing

RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA. The experiments consist of small RNA-seq and RNA-seq experiments from Naumovozyma castellii strains grown to mid-log phase. Two of the strains (WT_selection, xrn1_KO_selection) had hairpin-based silencing constructs against HIS3, URA3, and GFP genes and an extra copy of Argonaute (AGO1) and Dicer (DCR1) inserted into their genomes, which allowed this strain to be used in a genetic selection for components of budding-yeast RNAi pathways. Three other strains (WT_non-selection, xrn1_KO_non-selection, ago1_mut_non-selection) were in a genetic background in which their endogenous Argonaute protein was N-terminally FLAG-tagged (FLAG-tagged).

RNA干扰（RNA interference, RNAi）是一类基因沉默通路，可参与病毒防御、转座子沉默、异染色质形成及转录后基因沉默等生物学过程。酿酒酵母（Saccharomyces cerevisiae）中不存在该通路，但其他出芽酵母物种均保留了RNAi机制，例如卡氏纳瓦罗酵母（Naumovozyma castellii），该菌株拥有一套特殊的Dicer酶与经典Argonaute蛋白，二者均为基因沉默所必需。为鉴定出芽酵母RNAi通路中的其他作用因子，我们开展了无偏遗传筛选实验，最终将细胞质5'→3'外切核糖核酸酶Xrn1p鉴定为出芽酵母RNAi的辅助因子。在卡氏纳瓦罗酵母中敲除XRN1基因会显著削弱其基因沉默能力，该缺陷源于Xrn1p的多项核心功能：包括调控细胞内小干扰RNA（small interfering RNA, siRNA）的组分构成、影响siRNA加载至Argonaute蛋白的效率、降解切割产生的过客链，以及降解被切割的靶RNA。本数据集包含对数中期培养的卡氏纳瓦罗酵母菌株的小RNA测序与转录组测序数据：其中2株菌株（WT_selection、xrn1_KO_selection）携带针对HIS3、URA3及GFP基因的发夹结构沉默载体，且基因组中整合了额外拷贝的Argonaute（AGO1）与Dicer（DCR1）基因，可用于RNAi通路组分的遗传筛选；另外3株菌株（WT_non-selection、xrn1_KO_non-selection、ago1_mut_non-selection）的内源性Argonaute蛋白带有N端FLAG标签。