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Madhugiri R.,Institute of Microbiology and Molecular Biology | Pessi G.,ETH Zurich | Voss B.,Institute of Biology III | Hahn J.,Institute of Microbiology and Molecular Biology | And 6 more authors.
RNA Biology | Year: 2012

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D. © 2012 Landes Bioscience. Source

Witharana C.,Institute of Microbiology and Molecular Biology | Roppelt V.,Institute of Microbiology and Molecular Biology | Lochnit G.,Institute of Biochemistry | Klug G.,Institute of Microbiology and Molecular Biology | Evguenieva-Hackenberg E.,Institute of Microbiology and Molecular Biology
Biochimie | Year: 2012

The archaeal exosome is a protein complex involved in the degradation and the posttranscriptional tailing of RNA. The proteins Rrp41, Rrp42, Rrp4, Csl4 and DnaG are major subunits of the exosome in Sulfolobus solfataricus. In vitro, Rrp41 and Rrp42 form a catalytically active hexamer, to which an RNA-binding cap of Rrp4 and/or Csl4 is attached. Rrp4 confers strong poly(A) specificity to the exosome. The majority of Rrp41 and DnaG is detectable in the insoluble fraction and is localized at the cell periphery. The aim of this study was to analyze whether there are differences in the composition of the soluble and the insoluble exosomes. We found that the soluble exosome contains less DnaG and less Csl4 than the insoluble exosome which co-sediments with ribosomal subunits in sucrose density gradients. EF1-alpha was co-precipitated with the soluble exosome from S100 fractions using DnaG-directed antibodies, and from density gradient fractions using Rrp41-specific antibodies, strongly suggesting that EF1-alpha is an interaction partner of the soluble exosome. Furthermore, Csl4 was co-immunoprecipitated with the exosome using Rrp4-specific antibodies and vice versa, demonstrating the presence of heteromeric RNA-binding caps in vivo. To address the mechanism for poly(A) recognition by Rrp4, an exosome with an RNA-binding cap composed of truncated Rrp4 lacking the KH domain was reconstituted and analyzed. Although the deletion of the KH domain negatively influenced the degradation activity of the exosome, the poly(A) specificity was retained, showing that the KH domain is dispensable for the strong poly(A) preference of Rrp4. © 2011 Elsevier Masson SAS. All rights reserved. Source

Hou L.,Institute of Microbiology and Molecular Biology | Klug G.,Institute of Microbiology and Molecular Biology | Evguenieva-Hackenberg E.,Institute of Microbiology and Molecular Biology
Nucleic acids research | Year: 2014

The archaeal exosome is a phosphorolytic 3'-5' exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. Source

Hou L.,Institute of Microbiology and Molecular Biology | Klug G.,Institute of Microbiology and Molecular Biology | Evguenieva-Hackenberg E.,Institute of Microbiology and Molecular Biology
RNA Biology | Year: 2013

The archaeal RNA-degrading exosome contains a catalytically active hexameric core, an RNA-binding cap formed by Rrp4 and csl4 and the protein annotated as DnaG (bacterial type primase) with so-far-unknown functions in RNA metabolism. We found that the archaeal DnaG binds to the csl4-exosome but not to the Rrp4-exosome of Sulfolobus solfataricus. In vitro assays revealed that DnaG is a poly(A)-binding protein enhancing the degradation of adenine-rich transcripts by the csl4-exosome. DnaG is the second poly(A)-binding protein besides Rrp4 in the heteromeric, RNA-binding cap of the S. solfataricus exosome. This apparently reflects the need for effective and selective recruitment of adenine-rich RNAs to the exosome in the RNA metabolism of S. solfataricus. © 2013 Landes Bioscience. Source

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