Initiation of RNA Decay in Escherichia coli by 5′ Pyrophosphate Removal

Abstract

The common belief that endonucleolytic cleavage is the initial, rate-determining step of mRNA decay in Escherichia coli fails to explain the influence of 5' termini on the half-lives of primary transcripts. We have re-examined the initial events of RNA degradation in that organism by devising an assay to probe the 5' phosphorylation state of RNA and by employing a self-cleaving hammerhead ribozyme to investigate the degradative consequences of an unphosphorylated 5' end. These studies have identified a previously unrecognized prior step in decay that triggers subsequent internal cleavage by the endonuclease RNase E and thereby governs RNA longevity: the rate-determining conversion of a triphosphorylated to a monophosphorylated 5' terminus. Our findings redefine the role of RNase E in RNA degradation and explain how unpaired 5'-terminal nucleotides can facilitate access to internal cleavage sites within primary transcripts. Moreover, these results reveal a striking parallel between the mechanisms of mRNA decay in prokaryotic and eukaryotic organisms.

Specificity of RppH-dependent RNA degradation in Bacillus subtilis

“…However, we have recently described an alternative degradation pathway in B. subtilis in which the decay of primary transcripts is triggered by the RNA pyrophosphohydrolase RppH (previously known as YtkD), a Nudix hydrolase that removes two of the three 5′-terminal phosphates to generate a full-length intermediate that is monophosphorylated and therefore vulnerable to rapid 5′-exonucleolytic digestion by RNase J (7). A related 5′-end-dependent pathway is also present in bacterial species that lack RNase J, such as E. coli, where pyrophosphate removal triggers rapid cleavage by RNase E, the endonucleolytic activity of which is stimulated by a 5′ monophosphate (8)(9)(10).…”

The end defines the means in bacterial mRNA decay

“…In eukaryotes the distal phosphate is replaced by an inverted, methylated GMP to form the m 7 GpppX-cap; however, no such modification occurs in prokaryotes. The hydrolysis of the cap to a 5′ monophosphate is the first committed step in eukaryotic mRNA decay, and a paper by Celesnik et al 1 that appeared in the July 6, 2007 issue of Molecular Cell shows that a remarkably similar process occurs in E. coli.…”

“…Given that the 5′ end of bacterial mRNA is a 5′ triphosphate Celesnik et al 1 asked whether in E. coli there might be a previously unidentified step that triggers RNase E cleavage by converting the 5′ terminus to a monophosphate. To address this they developed a splinted ligation assay in which an antisense DNA oligonucleotide that extends past the RNA 5′ end is used to position another oligonucleotide (oligo X) adjacent to 5′ most nucleotide.…”

“…Much as the discovery of decapping led to new discoveries in eukaryotic mRNA decay, the results of this study are likely to generate new insights into prokaryotic decay for some time to come. Most bacterial mRNAs begin with a 5′ triphosphate and end with a stem-loop structure, and decay starts with endonuclease cleavage by RNase E. The preferred substrate for RNase E is RNA with a 5′ monophosphate, a property that is determined by the presence of a monophosphate-binding pocket within the catalytic domain of the enzyme, and Celesnik et al 1 describe a previously unknown step in mRNA decay in which pyrophosphate is removed from the 5′ end to generate a 5′ monophosphate substrate for cleavage by RNase E. This is the rate limiting step in decay, and subsequent cleavage by RNase E generates an upstream product with a 3′ hydroxyl that is degraded by 3′-5′ exonucleases and a downstream product with a 5′ monophosphate. This cycle is then repeated to complete degradation of the mRNA.…”

mRNA degradation and maturation in prokaryotes: the global players

“…The recently discovered 59 pyrophophatase RppH can convert the 59 PPP moiety of primary transcripts to a 59 monophosphate (62,63), an activity exploited by RNase E. In this novel decay pathway, internal cleavage by RNase E, is thus triggered by a prior event at the 59 end. The action of RppH and the recognition of the 59 P end by RNase E both require single-stranded 59 termini.…”