Transcriptome-wide Analysis of Exosome Targets in the Budding Yeast Saccharomyces cerevisiae

Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

研究目的：外泌体在RNA加工与监控过程中发挥核心作用，但目前其体内靶标范围与底物获取机制仍不明晰。我们采用了体内交联偶联深度测序技术（cross-linking coupled with deep sequencing，CRAC），可在活细胞中捕获酵母外泌体各亚基与其靶标之间的全转录组水平互作。
实验方法：我们对带有HTP标签的酵母外泌体蛋白开展CRAC实验，其中HTP标签的组成为His6 - TEV酶切位点 - 两段蛋白A的Z结构域，涉及的蛋白包括两种核酸酶亚基（Rrp44、Rrp6）以及两种结构亚基（Rrp41、Csl4）。每组实验至少独立重复两次，并分别进行分析。我们针对野生型（wild-type，WT）Rrp44以及两种催化突变体rrp44-endo（D91N、E120Q、D171N、D198N）和rrp44-exo（D551N）实施了CRAC实验。此外，我们还开发了基于可切割蛋白的split-CRAC技术，用于比对Rrp44的核酸内切酶与核酸外切酶靶标。为split-CRAC设计的质粒在RRP44开放阅读框（Open Reading Frame，ORF）的第241与242位氨基酸之间插入了PreScission蛋白酶酶切位点（PreScission protease cleavage site，PP），以便对纯化后的蛋白进行体外切割，同时携带His6标签以筛选对应的切割片段。
实验结果：对野生型Rrp44及其催化突变体的分析显示，CUT类与SUT类非编码RNA、核仁小RNA（small nucleolar RNAs，snoRNAs），以及最具代表性的转运RNA前体（precursor transfer RNAs，pre-tRNAs）和其他RNA聚合酶III（RNA polymerase III，Pol III）转录本，均被靶向进行寡腺苷酸化并经外泌体降解。未剪接的前体mRNA也被鉴定为Rrp44与Rrp6的靶标。基于可切割蛋白的split-CRAC实验结果表明，Rrp44的核酸内切酶与核酸外切酶活性在多数底物上存在协同作用。对寡腺苷酸化测序读段（sequencing reads，reads）的定位分析显示，核酸内切酶活性可能会释放停滞的外泌体底物。Rrp6优先结合具有二级结构的靶标，而这类靶标通常不与核心外泌体结合。这表明底物可通过多条途径抵达核酸酶。
研究结论：本研究首次绘制了酵母外泌体核酸酶复合物底物的全转录组图谱，并鉴定了野生型与突变型酵母细胞中外泌体各亚基的靶标。