Function and structure of the RNA-processing exosome in the hyperthermophilic archaeon Sulfolobus solfataricus

RNA的加工和降解是所有生物体的基本过程。在真核生物中，外泌体首先被确定为加工和降解RNA所必需的蛋白质复合物。通过在其底物的3′端添加短聚(A)尾部来激活其降解。我们正在研究古细菌外泌体，该外泌体磷酸化降解RNA，并通过rndp逆反应合成富a尾部。外泌体的保守的9个亚基核心是由亚基Rrp41和Rrp42组成的六聚体环，与rna结合蛋白Rrp4和Csl4的三聚体帽结合。古细菌外泌体的六聚体具有催化活性，而真核生物外泌体的六聚体则没有。古细菌外体含有古细菌特异性亚基，标记为细菌型引物酶DnaG。我们分析了超嗜热菌Sulfolobus solfataricus外泌体的RNA结合帽，发现i)同时含有Rrp4和Csl4的异质RNA结合帽存在ii) Rrp4而不是Csl4赋予外泌体poly(A)结合的优先性；iii) DnaG需要Csl4与外泌体相互作用；iv) DnaG包含一个新的RNA结合域，显示poly(a)偏好，并与Rrp4一起支持外泌体与富含a的RNA的相互作用；v) DnaG使外泌体能够向rRNA添加富a尾部；6)富a尾部增强了外泌体对rRNA的降解。我们的数据有力地表明，在S. solfataricus中，外显体对富含a的rna的选择性结合是重要的，并且DnaG参与了外显体对稳定rna的质量控制和/或适应环境变化。我们还发现rna结合蛋白Nop5通过Rrp4与外泌体相互作用。含有DnaG或nop的外泌体的空间结构尚不清楚。在本项目中，我们旨在分析古细菌和真核生物外泌体聚(A)偏好的分子基础。此外，优选的基因组编码的古菌外泌体底物应该在全球范围内进行鉴定。该项目的另一个目标是研究DnaG和rRNA之间的相互作用，重点研究含有DnaG的外泌体成功尾随rRNA所需的RNA决定因子。我们将尝试产生dnaG突变体，并在体内分析dnaG在磺胺虫RNA代谢中的作用。最后，通过重构蛋白复合物的SPEM分析，揭示含有DnaG或Nop5的外泌体的空间结构。我们的数据将揭示选择性RNA降解过程中聚(A)偏好的分子机制，这似乎在生命的所有领域都是保守的。古细菌稳定RNA的质量控制和转录后基因调控的机制，以及古细菌外泌体的结构将被阐明。因此，该项目的结果将有助于理解RNA加工和降解重要过程中涉及的机制。

RNA processing and degradation are essential processes in all living organisms. The exosome was first identified as an essential protein complex necessary for processing and degradation of RNA in Eukarya. It is activated for degradation by the addition of short poly(A) tails to the 3´-end of its substrates. We are investigating the archaeal exosome, which phosphorolytically degrades RNA and in a reverse reaction synthesizes A-rich tails using rNDPs. The conserved, nine-subunit core of the exosome is built of a hexameric ring of the subunits Rrp41 and Rrp42, to which a trimeric cap of the RNA-binding proteins Rrp4 and Csl4 is bound. The hexamer of the archaeal exosome but not of the eukaryotic exosome is catalytically active. The archaeal exosome harbours an archaea-specific subunit annotated as bacterial-type primase DnaG. We analysed the RNA-binding cap of the exosome in the hyperthermophile Sulfolobus solfataricus and found that i) heteromeric RNA binding caps containing both Rrp4 and Csl4 exist ii) Rrp4 but not Csl4 confers poly(A) binding preference to the exosome; iii) DnaG needs Csl4 for interaction with the exosome; iv) DnaG contains a novel RNA-binding domain, shows poly(A) preference and together with Rrp4 supports the interaction of the exosome with A-rich RNA; v) DnaG enables the addition of A-rich tails to rRNA by the exosome; vi) A-rich tails enhance the degradation of rRNA by the exosome. Our data strongly suggest that the selective binding of A-rich RNAs by the exosome is important in S. solfataricus and that DnaG is involved in quality control of stable RNAs by the exosome and/or in adaptation to environmental changes. We also found that the RNA-binding protein Nop5 interacts with the exosome through Rrp4. The spatial structure of DnaG- or Nop-containing exosomes is not known. In the proposed project we aim to analyse the molecular basis of the poly(A) preference of the exosome in Archaea and Eukarya. Furthermore, preferred, genome-encoded substrates of the archaeal exosome should be identified on a global scale. Another goal of the project is to study the interaction between DnaG and rRNA with focus on RNA determinants needed for successful tailing of rRNA by the DnaG-containing exosome. We will attempt to generate dnaG mutants and to analyse in vivo the role of DnaG in RNA metabolism of Sulfolobus. Finally, the spatial structure of the exosome containing DnaG or Nop5 should be uncovered by SPEM analysis of reconstituted protein complexes. Our data will unravel molecular mechanisms for poly(A) preference in course of selective RNA degradation, which seem to be conserved in all domains of life. The mechanisms for quality control of stable RNA and posttranscriptional gene regulation in Archaea, and the structure of the archaeal exosome will be elucidated. The results from this project will thus contribute to the understanding of the mechanisms involved in the important processes of RNA processing and degradation.