RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli

The control of mRNA stability is an important component of regulation in bacteria. Processing and degradation of mRNAs are initiated by an endonucleolytic attack, and the cleavage products are processively degraded by exoribonucleases. In many bacteria, these RNases, as well as RNA helicases and other proteins, are organized in a protein complex called the RNA degradosome. In Escherichia coli, the RNA degradosome is assembled around the essential endoribonuclease E. In Bacillus subtilis, the recently discovered essential endoribonuclease RNase Y is involved in the initiation of RNA degradation. Moreover, RNase Y interacts with other RNases, the RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase in a degradosome-like complex. In this work, we have studied the domain organization of RNase Y and the contribution of the domains to protein-protein interactions. We provide evidence for the physical interaction between RNase Y and the degradosome partners in vivo. We present experimental and bioinformatic data which indicate that the RNase Y contains significant regions of intrinsic disorder and discuss the possible functional implications of this finding. The localization of RNase Y in the membrane is essential both for the viability of B. subtilis and for all interactions that involve RNase Y. The results presented in this study provide novel evidence for the idea that RNase Y is the functional equivalent of RNase E, even though the two enzymes do not share any sequence similarity.     

RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue,RNase E

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half-lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne-1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half-lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.     

Re-expression of various i-antigens in Dileptus anser after temporary transformation of serotype

RNP particles containing 20S prosomes (alpha RNP) isolated from human epidermoid carcinoma cell line A-431 are shown to posses strong and regulated endonuclease activity specific for high-molecular-weight RNA, particularly, specific mRNAs. Furthermore, alpha-RNP destabilize the 3'-untranslated regions of c-myc mRNA, creating a specific cleavage pattern. Cleavage point within Alu sequence in high-molecular-weight RNA has been localized by primer-extension method. This RNase activity is induced under the action of EGF. alpha-RNP involvement in the coordinated control of processing and stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of alpha-RNP can represent a link between EGF signalling pathway and RNA processing and degradation.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

The RNase E/G-type endoribonuclease of higher plants is located in the chloroplast and cleaves RNA similarly to the E. coli enzyme

RNase E is an endoribonuclease that has been studied primarily in Escherichia coli, where it is prominently involved in the processing and degradation of RNA. Homologs of bacterial RNase E are encoded in the nuclear genome of higher plants. RNA degradation in the chloroplast, an organelle that originated from a prokaryote similar to cyanobacteria, occurs via the polyadenylation-assisted degradation pathway. In E. coli, this process is probably initiated with the removal of 5'-end phosphates followed by endonucleolytic cleavage by RNase E. The plant homolog has been proposed to function in a similar way in the chloroplast. Here we show that RNase E of Arabidopsis is located in the soluble fraction of the chloroplast as a high molecular weight complex. In order to characterize its endonucleolytic activity, Arabidopsis RNase E was expressed in bacteria and analyzed. Similar to its E. coli counterpart, the endonucleolytic activity of the Arabidopsis enzyme depends on the number of phosphates at the 5' end, is inhibited by structured RNA, and preferentially cleaves A/U-rich sequences. The enzyme forms an oligomeric complex of approximately 680 kDa. The chloroplast localization and the similarity in the two enzymes' characteristics suggest that plant RNase E participates in the initial endonucleolytic cleavage of the polyadenylation-stimulated RNA degradation process in the chloroplast, perhaps in collaboration with the two other chloroplast endonucleases, RNase J and CSP41.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.