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

Abstract

The best characterized pathway for the initiation of mRNA degradation in Escherichia coli involves the removal of the 5'-terminal pyrophosphate to generate a monophosphate group that stimulates endonucleolytic cleavage by RNase E. We show here however, using well-characterized oligonucleotide substrates and mRNA transcripts, that RNase E can cleave certain RNAs rapidly without requiring a 5'-monophosphorylated end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorized as 'direct' or 'internal' entry, appears to be multiple single-stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has alluded to the existence of a 5' end-independent mechanism of mRNA degradation, the relative simplicity of the requirements identified here for direct entry suggests that it could represent a major means by which mRNA degradation is initiated in E. coli and other organisms that contain homologues of RNase E. Our results have implications for the interplay of translation and mRNA degradation and models of gene regulation by small non-coding RNAs.

Fig. 1

Cleavage of 5′-hydroxylated oligonucleotide substrate by the N-terminal half of RNase E. A. The cleavage of 5′-hydroxylated BR13 (McDowall et al., 1995; Tock et al., 2000) labelled at the 3′ end with fluorescein. The reaction products were separated on a 15% polyacrylamide, sequencing-type gel. Lanes 1–6 contain samples taken 0, 2, 5, 10, 50 and 120 min after mixing substrate and enzyme. The reaction components were stored on ice prior to mixing. The position of bands corresponding to substrate (S) and downstream product (P) are indicated on the right of the panel. The enzyme and substrate concentrations at the start of a reaction were 5 and 250 nM respectively. B. Plots of the amount of product formed with time for BR13 that has a hydroxyl (open circles) or monophosphate (closed circles) group at its 5′ end. The conditions for cleavage of the latter were as (A). The lines with short and long dashes represent linear fits of data for the 5′-hydroxylated substrate between 0 and 5 min and 10 and 120 min respectively. C. Data for assays to which either fresh substrate (solid line) or enzyme (dotted line) was added after the reactions had reached the second, slower phase. The concentrations of enzyme and substrate at the start of the reaction were as described for (A). The addition of a second aliquot of enzyme or substrate (one-quarter volume) altered the total concentrations added to 8 nM enzyme and 200 nM substrate or 4 nM enzyme and 400 nM substrate respectively. D. Data for the cleavage of 5′-hydroxylated BR13 at an initial concentration of 250 nM by NTH-RNase E at concentrations of 1.25 nM (white circles), 2.5 nM (light grey), 5 nM (dark grey) or 25 nM (black). The vertical line with short dashes indicates the time point after which cleavage is judged to be slow.

Fig. 2

Preincubation at the reaction temperature abolishes the rapid phase of cleavage of BR13. The figure shows data for two cleavage assays using 5′-hydroxylated BR13 and RNase E at concentrations of 250 and 5 nM respectively. Open and closed circles represent data for substrate that had been stored on ice or prewarmed to the reaction temperature respectively, before combining with enzyme.

Fig. 3

Analysis of 5′-hydroxylated BR13 using CD and enzymatic assays. A. A schematic drawing of a parallel quadruplex. B. The arrangement of four guanosines in a G-quartet. Dashed lines represent the eight hydrogen bonds formed between the four guanosines. A cation (e.g. potassium) is usually located in the middle of two quartets and is shown here as a black sphere. Taken from Burge et al. (2006). C. The CD spectra of BR13 (grey lines) and LU13 (black lines) at a concentration of 7.5 μM in 25 mM bis-Tris-Propane (pH 8.3) and 100 mM NaCl. The solid and dotted line represent data collected at 4°C and 37°C respectively. LU13 is a BR13 derivative that has the central G of the 5′ triplet replaced with an A. D. Data for the cleavage of 5′-hydroxylated BR13 (open circles) and LU13 (closed circles) as per the conditions described in Fig. 1.

Fig. 4

Assaying the cleavage of single-stranded segments linked via conjugation. A. The binding of 5′-biotinylated LU13 to streptavidin as monitored using native gel electrophoresis. The position of streptavidin was detected by staining with Coomassie blue. Labelling on the right indicates the position of unbound streptavidin (U) and streptavidin bound to 5′-biotinylated LU13 (B). Lanes 1–4 correspond to oligonucleotide amounts of 0.3, 0.6, 1.2 and 1.5 nmol respectively. The amount of streptavidin was 0.15 nmol in all the conjugation reactions. Lane S contains only streptavidin. 5′-hydroxylated LU13 was included as a control. B. The results of incubating 5′-biotinylated (Biotin) and hydroxylated (HO) LU13 with NTH-RNase E after mixing a fourfold molar excess with streptavidin (A). Lanes 1–5 correspond to samples removed after incubating with enzyme for 0, 5, 15, 30 and 60 min. The enzyme and substrate concentrations were 5 and 65 nM respectively. Lanes 6 and 7 correspond to samples incubated in reaction buffer without enzyme for 0 and 60 min respectively. The samples were separated on a denaturing 15% (w/v) polyacrylamide gel. C. Plots of the amount of product formed with time for the reactions shown in (B) and controls that were not mixed with streptavidin prior to incubating with NTH-RNase E (original gels not shown). Open and closed circles correspond to 5′-biotinylated and hydroxylated LU13, respectively, which had been preincubated with streptavidin, while the open and closed squares correspond to incubation of these substrates directly with NTH-RNase E. Closed triangles correspond to prewarmed 5′-monophosphorylated BR13. The substrate concentration was reduced to 65 nM (cf. Fig. 1) to slow the reaction, thereby permitting easier comparison with the 5′-monophosphorylated BR13 control.

Fig. 5

Quadruplexed RNA is bound with higher affinity. A. A schematic of quadruplex BR13 binding to a principal dimer of RNase E (top view). The two RNase E protomers that form the principal dimer are shown in dark and lighter grey. The 5′ sensor and the RNA-binding channel that leads to the catalytic site formed at the protomer–protomer interface are drawn as white octagons and rectangles respectively. The stacked G-quartets and nucleotides of the single-stranded tails of quadruplexed BR13 are represented by a transparent block and beads respectively. Two of the four single-stranded tails can bind the two RNA-binding channels of a principal dimer, and be cleaved at the active sites located at the interface between the protomers. The nucleotides that form the 3′ product of cleavage are coloured grey. The schematic is drawn to scale. Arrows indicate the width (10 nm) and depth (5 nm) of the principal dimer. B. The Michaelis–Menten analysis of quadruplexed, 5′-hydroxylated BR13 (_quadHOBR13-Fl). Data obtained during cleavage assays were transformed into an Eadie-Hofstee plot. A linear fit to the data points is shown as a black line. This substrate was assayed over a concentration range of 25 nM to 4.0 μM using NTH-RNase E at a fixed concentration of 1 nM. The negative slope and the y-intercept of this plot represent the K_M and k_cat respectively. C. A Michaelis–Menten analysis for the cleavage of 5′-hydroxylated BR13 (_HOBR13-Fl). Data are plotted as the initial rate (v) over substrate concentration (a). This substrate was assayed over a concentration range of 200 nM to 16 μM using 50 nM of NTH-RNase E. D. An Eadie-Hofstee plot for the cleavage of prewarmed 5′-monophosphorylated BR13 (_PBR13-Fl). This substrate was assayed over a concentration range of 50 nM to 6 μM using 1 nM of NTH-RNase E.

Fig. 6

Cleavage of cspA mRNA by NTH-RNase E. A. A schematic diagram of the cspA gene. The vertical arrow indicates the major RNase E cleavage site in the 3′ untranslated region of the mRNA (position +234 as determined by primer extension). The bent arrow and vertical line indicate the position of the site of transcriptional initiation and termination respectively. Adapted from Hankins et al. (2007). B. The results of incubating 5′-triphosphorylated RNAI and 9S RNA with NTH-RNase E at substrate and enzyme concentrations of 180 and 5 nM respectively. The position of substrate, which was generated by in vitro transcription, is labelled S on the right of each panel. Where generated to a detectable level the major product is labelled P. Lane 1 corresponds to substrate incubated without enzyme for 60 min, while lanes 2–6 correspond to substrate incubated with NTH-RNase E for 0, 5, 15, 30 and 60 min respectively. C. As (B), except that the substrate is cspA mRNA with a triphosphate or monophosphate at the 5′ end. D. As (B), except that the substrate is 5′-triphosphorylated cspA incubated with the T170V mutant, which is defective in 5′ end sensing (Jourdan and McDowall, 2008). The samples were separated on denaturing 7% (w/v) polyacrylamide, sequencing-type gels and visualized by staining with ethidium bromide.

Fig. 7

Cleavage of epd-pgk by NTH-RNase E. A and B. The results of incubating NTH-RNase E with 5′-triphosphorylated and 5′-monophosphorylated epd-pgk RNA respectively. C. The results of incubating 5′-triphosphorylated epd-pgk RNA with the T170V mutant of NTH-RNase E. The reaction conditions and labelling are as Fig. 6. The above samples were separated on denaturing 6% (w/v) polyacrylamide gels and visualized by staining with ethidium bromide. D. The position of the major site in 5′-triphosphorylated epd-pgk RNA cleaved by the T170V mutant. The stop and start codons of epd and pgk, respectively, are underlined and the intergenic region is in lower case. The positions are numbered according to the transcription initiation point of the P₀ promoter upstream of epd. This site was mapped by cloning and sequencing the 3′ fragment by RNA ligase-mediated reverse transcription PCR (Kime et al., 2008) and the 5′ fragment using standard reverse transcription and PCR (see Experimental procedures for details).

Fig. 8

Mapping of single-stranded regions in cspA mRNA recognized by NTH-RNase E. Single-stranded regions of cspA mRNA were mapped using NMIA modification data as constraints. A. A representative structure prediction for the region between nucleotides +159 and +268 that contains the major site of RNase E cleavage. The degree of NMIA modification of individual nucleotides is represented on a colour scale where red, orange, blue and grey represents high (50–100%), medium (25–49%), low (12–24%) and insignificant (0–11%) reactivity respectively. Nucleotides with reactivities between 25% and 100% were constrained to be single-stranded. The nucleotides for which no data were collected are represented by black letters. B, C and D. The normalized reactivity of each nucleotide position in three regions of cspA mRNA. Sites of cleavage that were detected after incubation with the D346N mutant of NTH-RNase E are indicated by closed triangles.