No abstract

Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate, creating a better substrate for subsequent ribonuclease digestion. For example, in Bacillus subtilis and related organisms, removal of the gamma and beta phosphates of primary transcripts by the RNA pyrophosphohydrolase RppH triggers rapid 5′-exonucleolytic degradation by RNase J. However, the basis for the selective targeting of a subset of cellular RNAs by this pathway has remained largely unknown. Here we report that purified B. subtilis RppH requires at least two unpaired nucleotides at the 5′ end of its RNA substrates and prefers three or more. The second of these 5′-terminal nucleotides must be G, whereas a less strict preference for a purine is evident at the third position, and A is slightly favored over G at the first position. The same sequence requirements are observed for RppH-dependent mRNA degradation in B. subtilis cells. By contrast, a parallel pathway for 5′-end-dependent RNA degradation in that species appears to involve an alternative phosphate-removing enzyme that is relatively insensitive to sequence variation at the first three positions.O f the mechanisms that control gene expression in all living organisms, mRNA degradation is among the least well understood. Within the same cell, the half-lives of distinct mRNAs can differ by up to two orders of magnitude (seconds to an hour in bacteria, minutes to more than a day in higher eukaryotes), with proportionate effects on mRNA levels (1). In addition, the lifetimes of many messages can be modulated in response to environmental signals.Although it was originally assumed that Escherichia coli could serve as a paradigm for mRNA degradation in all bacteria, it is now clear that many bacterial species use a different set of ribonucleases and distinct mechanisms to degrade mRNA. A gamma proteobacterium, E. coli generally relies on the essential endonuclease RNase E to cleave transcripts internally, generating RNA fragments that are then degraded to mononucleotides by a combination of further RNase E cleavage and 3′-exonuclease digestion (1). Remarkably, despite its crucial role in E. coli, RNase E is entirely absent from a large number of other bacteria, including many Gram-positive species and even some Gram-negative proteobacteria (2). Instead, those species generally contain one or more ribonucleases that are absent from E. coli. For example, Bacillus subtilis, Staphylococcus aureus, and Helicobacter pylori contain two such ribonucleases-RNase Y, a membraneassociated endonuclease, and RNase J, a 5′-monophosphatedependent 5′ exonuclease that is also capable of acting as an endonuclease-in addition to a number of 3′ exonucleases (2-6).Because primary transcripts in B. subtilis are protected from exonucleolytic degradation by their 5′-terminal triphosphate and 3′-terminal stem loop, it was initially believed that message degradation in that species bypasses those termini and begins with endonucleolytic cleavage, generating RNA fragments with un...

Highlights d Pancreatic cells exhibit lower phosphofructokinase activity than other cell types d High glucose elevates O-GlcNAcylation and genomic alterations in pancreatic cells d Reduction of RNR activity leads to nucleotide pool imbalance and KRAS mutations d PFK activity alters the sensitivity to high-glucose-induced genomic effects

Background:In bacteria, RppH triggers mRNA degradation by converting the 5Ј-terminal triphosphate to a monophosphate. Results: The substrate specificity of E. coli RppH has been determined, along with residues important for this property and species in which they are conserved. Conclusion:The specificity of RppH is species-dependent. Significance: These findings help to explain the selective influence of RppH on rates of mRNA decay.

SUMMARY Enterohemorrhagic E. coli harbors a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.