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

• Applicant: Professorin Dr. Gabriele Klug
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2011 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 211453750

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.

• DFG Programme: Research Grants
• Co-Investigator: Professorin Dr. Elena Evguenieva-Hackenberg