Institute of Microbiology and Molecular Biology, Justus-Liebig-University, 35392, Giessen, Germany.
Institute of Bioinformatics and Systems Biology, Justus-Liebig-University, 35392, Giessen, Germany.
BMC Genomics. 2020 Nov 16;21(1):797. doi: 10.1186/s12864-020-07200-x.
The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging.
To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected.
In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.
古菌 exosome 是一种 exoribonucleolytic 多蛋白复合物，可从 3'到 5'方向对单链 RNA 进行磷酸解降解。在反转反应中，它可以在 RNA 的 3'端添加富含 A 的尾巴。exosome 的催化中心位于其六聚体核心的 aRrp41 亚基中。其 RNA 结合亚基 aRrp4 和 aDnaG 赋予复合物聚 A 偏好。古菌 exosome 在体外得到了深入研究，但对其在细胞中与天然底物的相互作用仍知之甚少，特别是因为分析 exoribonuclease 与 RNA 的全转录组相互作用具有挑战性。
为了在全局范围内确定 exosome 与 RNA 的结合位点，我们使用针对 chrenarchaeon Sulfolobus solfataricus 的 aRrp4 和 aRrp41 的抗体进行了单个核苷酸分辨率的 UV 交联和免疫沉淀 (iCLIP) 分析。获得的 cDNA 读段中相当大的比例（17-19%）无法映射到基因组。相反，它们对应于腺嘌呤丰富的 RNA 尾巴，这些尾巴是由 exosome 转录后合成的，并且对应于环状 RNA（circRNA）。我们鉴定了对应于两个同源转座酶相关 mRNA 5'部分的新型 circRNA。为了检测 exosome 的首选底物，将 iCLIP 读段与使用 RNA-Seq 数据获得的转录丰度进行比较。在强烈富集的 exosome 底物中，有与 tRNA 反义、重叠 3'UTR 和含有聚 A 延伸的 RNA。大多数读段计数和交联位点映射在 mRNAs 中。此外，还检测到 RNA 5'端聚类的意外交联位点。
在这项研究中，通过 iCLIP 分析了 exoribonuclease 的 RNA 靶标。这些数据证明了古菌 exosome 作为一种与所有 RNA 类相互作用的 exoribonuclease 和 RNA 加尾酶的作用，并强调了其在 mRNA 周转中的作用，这对于原核细胞适应不断变化的环境条件非常重要。基因 5'端附近交联位点的聚类表明，S. solfataricus exosome 同时结合 RNA 的两个末端。这可能有助于防止专门用于 3'-5'方向降解的 mRNAs 的翻译。
