Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation

Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3 end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways. RNA 3'end seq of early (10min 4sU labelled) pA+ and pA+ + pA- RNA 3' ends after rapid depletion of exosome core- (EXOSC3) and co-factors (ZCCHC8, ZFC3H1) in OsTIR1-expressing HeLa cells.

哺乳动物多亚基RNA外泌体介导核质转录本的周转，该过程由两种衔接复合物介导：核外泌体靶向（Nuclear EXosome Targeting, NEXT）复合物与Poly(A)尾外泌体靶向（Poly(A) tail eXosome Targeting, PAXT）连接体。此前针对NEXT与PAXT的功能研究大多采用长期因子耗竭策略，这易引发间接表型的产生。本研究通过快速耗竭NEXT、PAXT及核心外泌体组分，揭示了其急性缺失所带来的直接效应。总体而言，蛋白质组变化幅度较小，且主要由其他外泌体及衔接蛋白亚基的共耗竭所主导，这反映了潜在的亚复合物组成模式。尽管对新合成RNA开展的平行高分辨率3'端测序验证了此前确立的因子特异性，但该结果同时表明，长期因子耗竭的测序数据集因次级效应出现结果虚高的问题。最为显著的是，在长期NEXT耗竭样本中观察到的普遍内含子降解表型，在短期耗竭样本中完全无法被检测到；这一结果反而凸显了NEXT对携带小核仁RNA（small nucleolar RNA, snoRNA）的内含子的靶向作用。对这些内含子的进一步分析揭示了一种罕见的、不依赖核心外泌体的RNA降解模式。本研究强调，RNA积累是长期降解因子耗竭的间接结果，我们推测这一现象至少部分源于替代性RNA降解通路的耗竭。本研究在表达OsTIR1的海拉（HeLa）细胞中，对外泌体核心组分（EXOSC3）及辅助因子（ZCCHC8、ZFC3H1）进行快速耗竭后，对早期（10分钟4-硫尿苷（4-thiouridine, 4sU）标记）的poly(A)+（pA+）RNA以及poly(A)+与poly(A)-（pA+ + pA-）RNA的3'端开展了高通量测序。