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

Abstract

RNase E, a central component involved in bacterial RNA metabolism, usually has a highly conserved N-terminal catalytic domain but an extremely divergent C-terminal domain. While the C-terminal domain of RNase E in Escherichia coli recruits other components to form an RNA degradation complex, it is unknown if a similar function can be found for RNase E in other organisms due to the divergent feature of this domain. Here, we provide evidence showing that RNase E forms a complex with another essential ribonuclease-the polynucleotide phosphorylase (PNPase)-in cyanobacteria, a group of ecologically important and phylogenetically ancient organisms. Sequence alignment for all cyanobacterial RNase E proteins revealed several conserved and variable subregions in their noncatalytic domains. One such subregion, an extremely conserved nonapeptide (RRRRRRSSA) located near the very end of RNase E, serves as the PNPase recognition site in both the filamentous cyanobacterium Anabaena PCC7120 and the unicellular cyanobacterium Synechocystis PCC6803. These results indicate that RNase E and PNPase form a ribonuclease complex via a common mechanism in cyanobacteria. The PNPase-recognition motif in cyanobacterial RNase E is distinct from those previously identified in Proteobacteria, implying a mechanism of coevolution for PNPase and RNase E in different organisms.

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803

“…This conclusion is similar to those from studies revealing similar responses to C-terminal deletion of a cold-induced RNA helicase, Lmo1722, which resulted in disassociation from the 50S ribosome in Listeria monocytogenes (38) and removal of CshA from the Staphylococcus aureus degradosome (14). While thylakoid membrane-associated polysomes have been reported in cyanobacteria (42), the subcellular localization of the cyanobacterial degradosome has not been determined (39). It is predicted that the Synechocystis degradosome is soluble since the RNaseE does not contain the C-terminal scaffolding domain required for membrane attachment (39), as observed in some bacteria (4,5,(12)(13)(14).…”

“…However, the mass spectrometric analysis of the polysome pellet also detected PNPase and RNase E degradosome components that correspond to the recently identified minimal degradosome in cyanobacteria (39). Interaction with an RNA helicase such as CrhR was not tested when it was shown previously that the Synechocystis RNase E and PNPase interact as a complex (39). Thus, while our data indicate that CrhR cosediments with polysome and degradosome components, we cannot determine if it directly interacts with the degradosome complex.…”

“…The data therefore suggest that CrhR is associated with translation, consistent with the proteomic analysis of the polysome pellet. However, the mass spectrometric analysis of the polysome pellet also detected PNPase and RNase E degradosome components that correspond to the recently identified minimal degradosome in cyanobacteria (39). Interaction with an RNA helicase such as CrhR was not tested when it was shown previously that the Synechocystis RNase E and PNPase interact as a complex (39).…”

“…Although other studies investigating Synechocystis subcellular localization failed to detect CrhR, the lack of CrhR detection in these studies most likely resulted from growth conditions that did not induce abundant CrhR expression, as the cultures were grown at 30 and 34°C, temperatures at which CrhR is expressed at a basal level (24). Results of those studies included the lack of detection of a DEAD box RNA helicase in the Synechocystis minimal degradosome (39) and membrane (37,(43)(44)(45) or cytoplasmic (46) extracts.…”

“…While thylakoid membrane-associated polysomes have been reported in cyanobacteria (42), the subcellular localization of the cyanobacterial degradosome has not been determined (39). It is predicted that the Synechocystis degradosome is soluble since the RNaseE does not contain the C-terminal scaffolding domain required for membrane attachment (39), as observed in some bacteria (4,5,(12)(13)(14). Although other studies investigating Synechocystis subcellular localization failed to detect CrhR, the lack of CrhR detection in these studies most likely resulted from growth conditions that did not induce abundant CrhR expression, as the cultures were grown at 30 and 34°C, temperatures at which CrhR is expressed at a basal level (24).…”