The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex

Abstract

SummaryIn most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein-protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.

DEAD-Box RNA Helicases in Gram-Positive RNA Decay

“…Very recently, bacterial two-hybrid analysis in B. subtilis and S. aureus identified the CshA RNA helicase as a potential member of a degradosome in Gram-positive bacteria (Lehnik-Habrink et al, 2010;Roux et al, 2011). These results are nicely consistent with data from our laboratory that show that a mutation in cshA results in a stabilization of the agr mRNA (Oun et al, in preparation).…”

5′ End-independent RNase J1 Endonuclease Cleavage of Bacillus subtilis Model RNA

“…trp leader RNA and 5Ј-SSS RNA were synthesized to contain a labeled triphosphate 5Ј end, which would leave the RNA sensitive to RNase J1 endonuclease activity but resistant to 5Ј-exonuclease activity. To increase the yield of in vitro transcription, which is limited because of the low concentration of initiating nucleotide needed to get significant incorporation of [␥- 32 P]NTP at the 5Ј end, two (for 5Ј-SSS RNA) or three (for trp leader RNA) guanosine residues were added to the 5Ј end. Incubation of trp leader RNA with RNase J1 resulted in a gradual disappearance of full-length substrate (140 nt) and appearance of a ϳ100-nt band that was the 5Ј product of cleavage at nt 101 (Fig.…”

“…There are indications that RNase Y, like RNase E in E. coli, is a central element of an RNA degradosome complex that includes RNase J1 and PNPase (18,32). We investigated the effect of reducing RNase Y expression on trp leader RNA decay, using a strain in which RNase Y expression is under IPTG-inducible control and is grown in 1 mM ITPG, as above for the RNase J1 mutant strain.…”

“…Our hypothesis to explain the extreme stability of 5Ј-SSSϩNotI RNA in the RNase J1 mutant strain, which involves resistance to RNase J1 5Ј-exonuclease activity, cannot apply to RNase Y, as the latter enzyme has only endonuclease activity. Rather, the recent evidence that RNase Y exists in a degradosome complex that includes RNase J1 (18,32) may suggest that the effect of depleting RNase Y on trp leader RNA decay is an indirect one, resulting from a negative effect on RNase J1 activity. We believe that the close correlation of half-lives for all four constructs in the RNase J1 and RNase Y mutant strains lends credence to this hypothesis.…”

mRNA degradation and maturation in prokaryotes: the global players

“…The components identified by in vivo cross-linking and bacterial two-hybrid screening were enolase, phosphofructokinase, the RNA helicase CshA, PNPase and RNases J1/J2 (21,109). The observation that key nucleases, other than RNase E, can also form a degradosome, underlines the general importance of protein complexes for RNA maturation and decay.…”