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Acetylation regulates the oligomerization state and activity of RNase J, the major ribonuclease of Helicobacter pylori

ABSTRACT: In Helicobacter pylori, the major actor in post-transcriptional regulation is an RNA degradosome assembly composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe the oligomeric state and post-translational modifications of this protein complex. Our crystal structure of RNase J at 2.75 Å resolution reveals a homotetrameric quaternary state, and solution studies show that purified RNase J forms monomers and dimers, that RhpA is a monomer, and that the two proteins interact to form a stable complex with a 1:1 stoichiometry. Using mass spectrometry, we observed that RNase J is acetylated on multiple residues, one of which, K649, is important for RNase J oligomerization. Mutations targeting this residue and others affect the dimerization and in vitro activity of RNase J, suggesting that the dimer is the active form. In H. pylori, we observed that several of the identified acetylated residues impact on cell morphology, suggesting their importance for RNase J cellular function. Using H. pylori mutants, we found that several pathways contribute to RNase J acetylation. We propose acetylation as a regulatory level controlling RNase J properties and as a consequence the activity of the H. pylori RNA degradosome.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Helicobacter Pylori B8

SUBMITTER: Thibaut Douché

LAB HEAD: Hilde De Reuse

PROVIDER: PXD027670 | Pride | 2023-12-12

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20190624-Helicobacter_pilory_693745.fasta	Fasta
	20191223_Q2_TD_ColQ1-83_Tejada_Gel_AT1.raw	Raw
	20191223_Q2_TD_ColQ1-83_Tejada_Gel_AT2.raw	Raw
	20191223_Q2_TD_ColQ1-83_Tejada_Gel_AT3.raw	Raw
	20191223_Q2_TD_ColQ1-83_Tejada_Gel_AT4.raw	Raw

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RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Project description:Ribonucleases (RNases) are central actors in post-transcriptional regulation, a major level of regulation of gene expression in all cells. This control plays an important role in the bacterial pathogen Helicobacter pylori, although only the function of RNase J was characterized so far. Here, we studied the RNase R enzyme from H. pylori, a 3’-5’ exoribonuclease whose ortholog in Escherichia coli was reported to display, in addition, helicase activity and to be able to hydrolyze RNA substrates with double stranded structures. We observed that HpRNase R protein does not carry the domains responsible for helicase activity in E. coli and accordingly that the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The RNase R helicase domain loss is widespread among the Campylobacterota and occurred gradually during their evolution. Furthermore, an in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori, was discovered. Phylogenomics suggests that this interaction might occur in other bacteria of the phylum Campylobacterota. Purified RhpA facilitates the degradation of double stranded RNA substrates by HpRNase R, showing that this complex is functional. HpRNase R has a minor role of in 5S rRNA maturation and, as shown by RNA-Seq, few targets in H. pylori all of them being included in the RhpA regulon. In conclusion, we describe a new type of RNase R that lacks some of the features that were considered as hallmarks of RNase R proteins, but that has co-opted another RNA helicase, which we hypothesize helps it accomplish some of its functions in vivo.

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