Role of XRN2 ribonucleolytic activity in RNA metabolism

We analysed the effect of depriving the human cell of the catalytic activity of the nuclear 5’ to 3’ exoribonuclease XRN2. Catalytic amino acids in this protein had been defined previously, so it was possible to design a mutated catalytically inactive form of the protein (XRN2D233A-D235A) (PMID: 19194460). We created 293 Flp-In T-REx stable cell lines that induciby silence endogenous XRN2, and concomitantly express wild-type or inactive XRN2 in fusion with EGFP at the C-terminus. Thus, complementation of silencing of endogenous XRN2 with the expression of mutant version of the protein allows to directly link potential phenotypes with the lack of XRN2 enzymatic activity. To this end we isolated total RNA from tetracycline-treated cells, depleted it from rRNA and conducted strand-specific deep sequencing. 6 samples were analysed. 3 replicates of control cells (endogenous copy of XRN2 gene is silenced and catalytically active exogenous XRN2-EGFP is expressed) and 3 replicates of cells deprived of XRN2 ribonucleolytic activity (endogenous copy of XRN2 gene is silenced and catalytically inactive exogenous XRN2(D233AD235A)-EGFP is expressed)

本研究分析了剥夺人类细胞中细胞核5’→3’外切核糖核酸酶XRN2（nuclear 5’ to 3’ exoribonuclease XRN2）催化活性的效应。该蛋白的催化氨基酸已在既往研究中被明确，因此可设计该蛋白的催化失活突变体（XRN2D233A-D235A）（PMID: 19194460）。我们构建了293 Flp-In T-REx稳定细胞系，该细胞系可诱导性沉默内源性XRN2，并同时表达与增强绿色荧光蛋白（EGFP）在C端融合的野生型或失活型XRN2。因此，通过表达该蛋白的突变体以互补内源性XRN2沉默所产生的效应，可直接将潜在表型与XRN2酶活性的缺失直接关联。为此，我们从四环素处理的细胞中提取总RNA，去除其中的核糖体RNA（rRNA）并开展链特异性深度测序。本研究共分析6个样本：3个生物学重复的对照组细胞（内源性XRN2基因被沉默，同时表达催化活性的外源性XRN2-EGFP），以及3个生物学重复的丧失XRN2核糖核酸酶活性的细胞组（内源性XRN2基因被沉默，同时表达催化失活的外源性XRN2(D233AD235A)-EGFP）