The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components

Three novel components of the human exosome

Contains fulltext : 186951.pdf (publisher's version ) (Open Access)The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex

Antisense RNA protects mRNA from RNase E degradation by RNA–RNA duplex formation during phage infection

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg2+, pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA–mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression

Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur

RNase E and the High-Fidelity Orchestration of RNA Metabolism.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.Wellcome Trus

Rv0802c Acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule