The end defines the means in bacterial mRNA decay

Abstract

Bacterial mRNAs begin with a triphosphate on the first transcribed nucleotide, but the endonuclease long thought to initiate mRNA decay in E. coli (RNase E) only works well on RNA with a 5′ monophosphate. The first committed step in the degradation of mRNA in that organism now appears to be the conversion of the 5′ triphosphate to a monophosphate, a process that is functionally similar to mRNA decapping in eukaryotes.The 5′ end of newly synthesized RNAs bears a triphosphate derived from the first transcribed nucleotide. 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.In eukaryotes the process of mRNA decay generally involves loss of the 3′ poly(A) tail and removal of the cap by a decapping enzyme (Dcp2). The body of the mRNA is then degraded from the 5′ end by the 5′-3′ exonuclease Xrn1 and from the 3′ end by the exosome 2 . Prokaryotes such as E. coli use a distinctly different process in which mRNA is cleaved by the endonuclease RNase E to generate an upstream product with a 3′ hydroxyl and a downstream product with a 5′ monophosphate. The upstream product is rapidly cleared by 3′-5′ exonucleases and the 5′ monophosphate end of each downstream cleavage product activates subsequent rounds of endonuclease cleavage and exonuclease clearance (see bottom of Fig. 1). Thus, on the surface bacterial mRNA decay appears to be distinctly different from the major decay process in eukaryotes.The 5′ end of substrate mRNA dramatically affects its ability to be cleaved by RNase E, with the rate constant for 5′ monophosphate RNA more than an order of magnitude greater than that for RNA with a 5′ triphosphate or 5′ hydroxyl 3,4 . The basis for this 5′ end selectivity became clear from the crystal structure of the homotetrameric catalytic domain 5 , which showed the 5′ monophosphate end is hydrogen bonded into a pocket at the base of an RNA-binding channel. This interaction is thought to cause an allosteric change in the protein that juxtaposes a magnesium-coordinated hydroxyl with the scissile phosphate at a downstream site in the RNA substrate.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. DNA ligase will covalently join oligo X to the 5′ end of the RNA if it has a 5′ monophosphate, but not a 5′ hydroxl or 5′ triphosphate. By carefully optimizing reaction conditions and ...

Rapid cleavage of RNA by RNase E in the absence of 5′ monophosphate stimulation

“…Indeed, sufficient turnovers (∼20) occurred during a typical assay that the major upstream product was detectable by staining with ethidium bromide. Thus, in contrast to a recent commentary (Schoenberg, 2007), the presence of a 5′ monophosphate is not required for the rapid cleavage of all E. coli RNAs by RNase E. Consistent with this, the mRNA of cspA was still cleaved rapidly when incubated with the 5′ end‐sensing T170V mutant of NTH‐RNase E (Fig. 6D).…”

Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

“…In Escherichia coli , that process was thought to begin with endonucleolytic cleavage downstream of the 5′ end, most often by RNase E. Such cleavage would generate two fragments, both of which would be rapidly cleared by additional RNase Ecleavage and 3′-to-5′ exonuclease digestion (Schoenberg, 2007). …”

Complete genome sequence and annotation of the laboratory reference strainShigella flexneriserotype 5a M90T and genome-wide transcriptional start site determination

“…To determine transcriptional start sites, the RNA of three biological replicates in which the rRNA had been depleted with RiboZero (Illumina, Inc.) was used. To enrich for primary transcripts, we exploited the property that primary bacterial transcripts are protected from exonucleolytic degradation by their triphosphate (5'PPP) RNA ends [24], while RNAs containing a 5' monophosphate (5'P) are selectively degraded [16,24]. The rRNA-depleted RNA was split into two aliquots.…”

“…Primary transcripts of prokaryotes carry a triphosphate at their 5'-ends. In contrast, processed or degraded RNAs only carry a monophosphate at their 5'-ends [24]. The differential RNA-seq (dRNA-seq) approach used here exploits the properties of a 5'monophosphate-dependent exonuclease (TEX) to selectively degrade processed transcripts, thereby enriching for unprocessed RNA species carrying a native 5'-triphosphate [24].…”

“…In contrast, processed or degraded RNAs only carry a monophosphate at their 5'-ends [24]. The differential RNA-seq (dRNA-seq) approach used here exploits the properties of a 5'monophosphate-dependent exonuclease (TEX) to selectively degrade processed transcripts, thereby enriching for unprocessed RNA species carrying a native 5'-triphosphate [24]. TSSs can then be identified by comparing TEX-treated and untreated RNA-seq libraries, where TSSs appear as localized maxima in coverage enriched upon TEX-treatment [16].…”