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Structure and function of the archaeal exosome

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The RNA‐degrading exosome in archaea is structurally very similar to the nine‐subunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion‐dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3′ → 5′ direction. The archaeal nine‐subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH‐domain‐containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1‐domain‐containing subunits Rrp4 and Csl4 form an RNA‐binding trimeric cap on the top of the ring. In vivo, this cap contains Rrp4 and Csl4 in variable amounts. Rrp4 confers poly(A) specificity to the exosome, whereas Csl4 is involved in the interaction with the archaea‐specific subunit of the complex, the homolog of the bacterial primase DnaG. The archaeal DnaG is a highly conserved protein and its gene is present in all sequenced archaeal genomes, although the exosome was lost in halophilic archaea and some methanogens. In exosome‐containing archaea, DnaG is tightly associated with the exosome. It functions as an additional RNA‐binding subunit with poly(A) specificity in the reconstituted exosome of Sulfolobus solfataricus and enhances the degradation of adenine‐rich transcripts in vitro. Not only the RNA‐binding cap but also the hexameric Rrp41–Rrp42 ring alone shows substrate selectivity and prefers purines over pyrimidines. This implies a coevolution of the exosome and its RNA substrates resulting in 3′‐ends with different affinities to the exosome. WIREs RNA 2014, 5:623–635. doi: 10.1002/wrna.1234 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Processing > 3' End Processing RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms

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Core components of the archaeal exosome. (a) Exemplary silver‐stained sodium dodecyl sulfate‐polyacrylamide gel with the exosome purified from Sulfolobus solfataricus cell‐free extract. Rrp41‐directed serum was used for the coimmunoprecipitation (CoIP). M, marker proteins, the sizes in kDa are indicated. CoIP, elution fraction, the exosomal subunits are marked. (b) Schematic representation of the protein domains in the subunits of the archaeal exosome. RPD, RNase PH domain; N, N‐terminal domain; C, C‐terminal domain; S1, S1 domain; KH, KH domain; Zn, Zn‐ribbon domain. The asterisk marks the active RPD of Rrp41.
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Schematic representation of the structure of the archaeal exosome and its interaction with RNA. (a) Isoforms of the recombinant exosome. Alternating RPD‐containing subunits Rrp41 and Rrp42 (barrels marked with Rrp41 and Rrp42, respectively) are arranged in a hexameric ring. On the top of the ring, three S1‐domain‐containing subunits (ovals representing Rrp4 or Csl4) are located. DnaG binds to the Csl4‐exosome but not to the Rrp4‐exosome. Complexes with Rrp4–Csl4–DnaG–caps corresponding to the exosome in vivo can also be reconstituted. Rrp4 and Csl4 are present in different amounts in such complexes. (b) DnaG, an Rrp41, an Rrp42, and an Rrp4 subunit are removed to allow a view into the central channel of the hexameric ring and to the domains of the RNA‐binding cap. The nucleotides of an RNA substrate are indicated by gray circles. The first four nucleotides (as numbered from the 3′‐end of the substrate) are in the active site of an Rrp41 subunit. Pi and Mg2+ are needed for catalysis. N, N‐terminal domain; S1, S1 domain; KH, KH domain; Zn‐r., Zn‐ribbon domain. (c) Model of adenine‐rich RNA bound to the DnaG‐exosome with heteromeric RNA‐binding cap, which contains two different proteins with poly(A) preference, Rrp4, and DnaG. We propose that the DnaG and the S1 domain of Rrp4 efficiently and selectively interact with adenine‐rich stretches in natural substrates of the archaeal exosome.
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Schematic overview on RNA degradation in different archaeal groups. All archaea with sequenced genomes possess genes for cleavage and polyadenylation specificity factor (CPSF) homologs with putative exoribonucleolytic 5′ → 3′and/or endonucleolytic activities, but genes for putative 3′ → 5′ exoribonucleases are not present in all of them. Archaeal genera for which exosome, RNase R, and CPSF homologs were characterized as RNases in vitro are indicated. Question marks indicate activities which were not confirmed experimentally in the indicated genera.
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The exosome was lost in some phylogenetic lineages of archaea. All genome‐sequenced archaea harbor the dnaG gene. In the presented phylogenetic distribution, lineages or strains which do not possess genes for the core subunits of the exosome rrp41 and rrp42 are highlighted in bold and marked with an asterisk. The maximum‐likelihood tree is based on nearly full‐length 16S rRNA gene sequences. A total of 120 genome‐sequenced archaea were included in the analysis (see Supporting Information). The phylogenetic tree was generated in ARB release 5.2 using the ‘All‐Species Living Tree’ Project (LTP) ARB database release LTPs111 (February 2013). 16S rRNA gene sequences of genome‐sequenced strains not included in the LTP database were obtained from NCBI (http://www.ncbi.nlm.nih.gov/), aligned in the SILVA Incremental Aligner (SINA) version v1.2.11 and added to the LTP tree (maximum‐likelihood tree) using the parsimony quick add marked tool of ARB. Sequences of non‐genome‐sequenced archaea were removed from the tree. Bar, 0.10 nucleotide substitutions per site. Archaea with sequenced genomes were used for 16S rRNA‐based phylogenetic analysis.
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RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
RNA Processing > 3′ End Processing
RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms

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