DEAD Box RhlB RNA Helicase Physically Associates with Exoribonuclease PNPase to Degrade Double-stranded RNA Independent of the Degradosome-assembling Region of RNase E

Abstract

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.RNA metabolism is a complex process affecting the control of gene expression. In bacteria, a multicomponent ribonucleolytic complex termed the RNA degradosome (1-4) has been identified as playing an important role in the control of mRNA degradation (for recent reviews, see Refs. 5-11). The multicomponent complex consists of: the RNA endonuclease RNase E, whose activity is essential for Escherichia coli cell growth (12-14), RNA processing (15, 16), and degradation (17, 18); the 3Ј-5Ј exoribonuclease PNPase (19); RhlB RNA helicase (20); and enolase (21), an enzyme involved in the glycolytic pathway and other chaperonin proteins (3,22). Interestingly, in addition to mRNAs, highly structured, stable RNA fragments have also been found to be associated with RNA degradosome complexes (3, 23), which implies quality control by the RNA degradosome for the biogenesis of stable RNAs. Degradosome complexity and its cooperation with individual protein components acting on degradation-targeted RNA, in vivo, remains to be discovered.Various approaches revealing protein-protein interactions in the degradosome indicate that the C-terminal region of RNase E serves as a scaffold that directly binds PNPase, RhlB RNA helicase, and enolase (24,25). No other interactions among these component proteins have been detected (25) or reported. Recently, a mini-degradosome complex (26) containing the scaffold region (without the N-terminal enzymatic region of RNase E), RhlB RNA helicase, and PNPase was reconstituted in vitro. These experiments revealed a functional inte...

Global analysis of Escherichia coli RNA degradosome function using DNA microarrays

“…The work reported here indicates that the actions of RNase E, PNPase, enolase, and RhlB helicase are required for normal RNA turnover in E. coli, establishing a role for each of these degradosome components in mRNA decay. Our results further show that the effects of mutations in degradosome components vary with individual messages; they provide evidence that the assembled degradosome complex mediates the decay of some transcripts, whereas other transcripts are likely to be degraded independently of the complex, possibly by recently identified assembled subsets of degradosome enzymes (28). Collectively, our findings imply the existence of structural features or biochemical factors that distinguish cellular categories of mRNAs marked for degradation.…”

“…Thus, mutations in degradosome constituents may affect the actions of unassociated constituents as well the actions of proteins in assembled degradosomes. Consistent with the possibility that degradosome components may operate in other formats, RhlB helicase has been shown to interact with PNPase independently of RNase E (28).…”

Small RNA-mediated regulation at the level of transcript stability

“…While it is still unclear why SgrS action depends on membrane localization of the target, one likely explanation is that the RNA degradosome is associated with the cytoplasmic membrane. 54 Another model is that co-translational membrane localization decreases the translation efficiency of ptsG mRNA, which allows SgrS to compete with ribosomes for binding to ptsG.…”

RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

“…In addition to RNase E, the major components of the E. coli RNA degradosome also include the polynucleotide phosphorylase (PNPase), the DEAD box RNA helicase RhlB, and the glycolytic enzyme enolase (Carpousis et al 1994;Vanzo et al 1998). RNA degradation mediated by the RNA degradosome is a highly cooperative and efficient process: RNase E cuts single-stranded RNA substrates preferably at AU-rich sites, PNPase further converts the generated fragments into diphosphate mononucleotides through its 3 ′ -5 ′ phosphorolytic activity, and the RNA helicase RhlB unwinds structured RNAs to facilitate their turnover by RNase E and PNPase (Liou et al 2002). Enolase in the RNA degradosome does not seem to act on RNA directly.…”