Elena Evguenieva-Hackenberg

The archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3′ → 5′ exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 Å. The structure reveals a hexameric ring-like arrangement of three Rrp41–Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA–degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.

An exosome-like complex in Sulfolobus solfataricus

We present the first experimental evidence for the existence of an exosome‐like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co‐immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal‐RNA‐processing protein 41 (Rrp41) led to the purification of a 250‐kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cep1 synthetic lethality 4; an RNA‐binding protein and exosome subunit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA‐related function for the archaeal DnaG‐like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate‐dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.

RNA polyadenylation in Archaea : not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome‐like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S−50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG‐like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41‐Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41‐Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.

Bacterial ribosomal RNA in pieces

The exact knowledge on the ribosomal RNA (rRNA) structure is an important prerequisite for work  with rRNA sequences in bioinformatic analyses and in experimental research. Most available rRNA sequences of bacteria are based on gene sequences and on similarity analyses using Escherichia coli rRNA as a standard. Therefore, it is often overlooked that many bacteria harbour mature rRNA ‘in pieces’. In some cases, the processing steps during the fragmentation lead to the removal of rRNA segments that are usually found in the ribosome. In this review, the current knowledge on the mechanisms of rRNA fragmentation and on the occurrence of fragmented rRNA in bacteria is summarized, and the physiological implications of this phenomenon are discussed.

New aspects of RNA processing in prokaryotes

The pivotal role of posttranscriptional gene regulation is strongly underlined by genome-wide analyses showing strikingly low correlation between mRNA and protein levels in bacterial and archaeal cells. The stability of an mRNA and its availability for translation contribute to posttranscriptional gene regulation, and are determined by the following factors: i) the cell-specific set of ribonucleases and related proteins, ii) regulatory RNAs, and iii) the sequence and structural features of the RNA molecule itself. High-resolution analyses of whole prokaryotic transcriptomes allow comprehensive mapping of processed transcripts, detection of essentially all expressed regulatory RNAs, and monitoring of the global impact of ribonucleases and other processing factors. This opens new perspectives for the understanding of the molecular mechanisms responsible for mRNA decay in prokaryotes.

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5‐insertion in the RNase J‐encoding gene was compared to the wild‐type and a difference in the length of the 5.8S‐like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′‐end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.

Expression of small RNAs in Rhizobiales and protection of a small RNA and its degradation products by Hfq in Sinorhizobium meliloti

Regulatory RNA plays a pivotal role in the regulation of bacterial gene expression. Here, five small RNAs were studied in Sinorhizobium meliloti - SmrC15, SmrC16, Sra33, 6S and the signal recognition particle (SRP) RNA, which are conserved among at least seven different Rhizobium and Sinorhizobium species. The amount of SmrC16 decreased in stationary phase, while the other RNAs were up-regulated. The smallest changes, maximally 2-fold, were observed for 6S RNA. In the distantly related Bradyrhizobium japonicum, the amount of 6S RNA was not increased in stationary phase, suggesting some functional divergence in the roles of this molecule in Rhizobiales in comparison to Escherichia coli. Different decay rates were observed for SmrC15 and SmrC16 of S. meliloti upon rifampicin treatment, revealing posttranscriptional regulation during growth. The use of a Deltahfq mutant showed that Hfq protects full-length SmrC16 from degradation and stabilises its specific degradation products.

Homoserine Lactones Influence the Reaction of Plants to Rhizobia

Bacterial quorum sensing molecules not only grant the communication within bacterial communities, but also influence eukaryotic hosts. N-acyl-homoserine lactones (AHLs) produced by pathogenic or beneficial bacteria were shown to induce diverse reactions in animals and plants. In plants, the reaction to AHLs depends on the length of the lipid side chain. Here we investigated the impact of two bacteria on Arabidopsis thaliana, which usually enter a close symbiosis with plants from the Fabaceae (legumes) family and produce a long-chain AHL (Sinorhizobium meliloti) or a short-chain AHL (Rhizobium etli). We demonstrate that, similarly to the reaction to pure AHL molecules, the impact, which the inoculation with rhizosphere bacteria has on plants, depends on the type of the produced AHL. The inoculation with oxo-C14-HSL-producing S. meliloti strains enhanced plant resistance towards pathogenic bacteria, whereas the inoculation with an AttM lactonase-expressing S. meliloti strain did not. Inoculation with the oxo-C8-HSL-producing R. etli had no impact on the resistance, which is in agreement with our previous hypothesis. In addition, plants seem to influence the availability of AHLs in the rhizosphere. Taken together, this report provides new insights in the role of N-acyl-homoserine lactones in the inter-kingdom communication at the root surface.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

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.