PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins

In this report, we demonstrate that exonucleolytic turnover is much more important in the regulation of sRNA levels than was previously recognized. For the first time, PNPase is introduced as a major regulatory feature controlling the levels of the small noncoding RNAs MicA and RybB, which are required for the accurate expression of outer membrane proteins (OMPs). In the absence of PNPase, the pattern of OMPs is changed. In stationary phase, MicA RNA levels are increased in the PNPase mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein. This growth phase regulation represents a novel pathway of control. We have evaluated other ribonucleases in the control of MicA RNA, and we showed that degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. RybB was also destabilized by PNPase. This work highlights a new role for PNPase in the degradation of small noncoding RNAs and opens the way to evaluate striking similarities between bacteria and eukaryotes.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.     

The RNase E/G-type endoribonuclease of higher plants is located in the chloroplast and cleaves RNA similarly to the E. coli enzyme

RNase E is an endoribonuclease that has been studied primarily in Escherichia coli, where it is prominently involved in the processing and degradation of RNA. Homologs of bacterial RNase E are encoded in the nuclear genome of higher plants. RNA degradation in the chloroplast, an organelle that originated from a prokaryote similar to cyanobacteria, occurs via the polyadenylation-assisted degradation pathway. In E. coli, this process is probably initiated with the removal of 5'-end phosphates followed by endonucleolytic cleavage by RNase E. The plant homolog has been proposed to function in a similar way in the chloroplast. Here we show that RNase E of Arabidopsis is located in the soluble fraction of the chloroplast as a high molecular weight complex. In order to characterize its endonucleolytic activity, Arabidopsis RNase E was expressed in bacteria and analyzed. Similar to its E. coli counterpart, the endonucleolytic activity of the Arabidopsis enzyme depends on the number of phosphates at the 5' end, is inhibited by structured RNA, and preferentially cleaves A/U-rich sequences. The enzyme forms an oligomeric complex of approximately 680 kDa. The chloroplast localization and the similarity in the two enzymes' characteristics suggest that plant RNase E participates in the initial endonucleolytic cleavage of the polyadenylation-stimulated RNA degradation process in the chloroplast, perhaps in collaboration with the two other chloroplast endonucleases, RNase J and CSP41.     

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn’s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features.     

Circadian clock genes period and cycle regulate photoperiodic diapause in the bean bug Riptortus pedestris males

The photoperiodic response is crucial for many insects to adapt to seasonal changes in temperate regions. It was recently shown that the circadian clock genes period (per) and cycle (cyc) are involved in the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris females. Here, we investigated the involvement of per and cyc both in the circadian rhythm of cuticle deposition and in the photoperiodic diapause of R. pedestris males using RNA interference (RNAi). RNAi of per and cyc disrupted the cuticle deposition rhythm and resulted in distinct cuticle layers. RNAi of per induced development of the male reproductive organs even under diapause-inducing short-day conditions, whereas RNAi of cyc suppressed development of the reproductive organs even under diapause-averting long-day conditions. Thus, the present study suggests that the circadian clock operated by per and cyc governs photoperiodism of males as that of females.