Obstacles to Scanning by RNase E Govern Bacterial mRNA Lifetimes by Hindering Access to Distal Cleavage Sites

Abstract

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

Keywords: 5′ terminus; RNA decay; RNA processing; RNase G; SgrS; phosphosugar stress; ribonuclease; ribosome; uORF; yigL.

Figure 1.. Protection of downstream mRNA cleavage sites in E. coli by discontinuities that result from protein binding.

(A) 5′ UTR of reporter mRNAs. Arrows, cleavage sites; gray ring, TRAP 11-mer; white rectangle, beginning of the protein coding region; broad black lines, heteropolymeric (AC)₃ or (AC)_7.5 or homopolymeric (C)₁₅ spacers. The regions encompassing upstream sites A, B, and C and downstream sites W, X, and Y are identical in sequence. See also Figure S1. (B) Endonucleolytic cleavage of the 5′ UTR of AC3 in vivo and in vitro. Equal amounts of total cellular RNA from isogenic wild-type (WT), ΔrppH, or Δrng strains of E. coli containing AC3 mRNA were examined by Northern blotting to detect cleavage within the AC3 5′ UTR. Alternatively, monophosphorylated AC3 RNA synthesized by in vitro transcription was partially digested with purified N-RNase E (20 nM) or RNase G (80 nM) and analyzed similarly. The blots were probed with a radiolabeled oligonucleotide complementary to an AC3 segment downstream of site Z (Table S2). M, boundary marker between the upstream and downstream cleavage sites. (C) Cleavage within the 5′ UTR of reporter mRNAs in vivo. Equal amounts of total cellular RNA from isogenic wild-type (WT) and ΔrppH (Δ) strains of E. coli containing each reporter mRNA (TR8, TR11, TR8+C15, or TR11+AC7.5) and TRAP were analyzed by Northern blotting as in panel (B). See also Figure S1. (D) Relative abundance of 5′ UTR cleavage products in wild-type cells. The sum of the intensities of the bands in panel (C) resulting from cleavage upstream of the obstacle insertion site (A + B + C) was divided by the corresponding sum for cleavage downstream (V + W + X + Y + Z). Data from three biological replicates were used to calculate each mean and standard deviation. See also Figures S2 and S4. (E) Decay rates of reporter mRNAs and their upstream cleavage products in E. coli. Transcription was arrested by adding rifampicin to wild-type cells that contained AC3, TR8, or TR11 and TRAP, and equal amounts of total cellular RNA isolated at time intervals thereafter were examined by Northern blotting. See also Figures S1, S2, and S4. (F) Context independence of the effect of discontinuities on downstream cleavage. The TRAP-binding site of TR8 or TR11 was inserted between the two principal RNase E cleavage sites within the 5′ UTR of ompAΔhp (an ompA mRNA derivative bearing an unpaired 5′ end), and cleavage upstream versus downstream of the insertion site was compared in wild-type cells containing TRAP. Data from three biological replicates were used to calculate each mean and standard deviation. See also Figure S3. (G) Effect of structural discontinuities in the 5′ UTR on gene expression, β-galactosidase levels were compared in wild-type cells containing both TRAP and a lacZ translational fusion of AC3, TR8, or TR11 and normalized to AC3-lacZ. Data from three biological replicates were used to calculate each mean and standard deviation.

Figure 2.. Ability of a discontinuity to protect downstream sites from cleavage by purified N-RNase E.

Monophosphorylated (MonoP) or triphosphorylated (TriP) AC3, TR8, or TR11 RNAs that had been synthesized by in vitro transcription and mixed with TRAP were treated for 4 min with equal amounts of purified N-RNase E (20 nM). The reaction products were then analyzed by Northern blotting as in Figure 1. M, boundary marker between the upstream and downstream cleavage sites. See also Figures S2, S4, and S5.

Figure 3.. Effect of ribosome binding on downstream cleavage in E. coli.

(A) 5′ UTR of reporter mRNAs containing an upstream open reading frame. Arrows, cleavage sites; white rectangles, protein coding regions. (B) Relative abundance of 5′ UTR cleavage products for SD-low, SD-med, SD-high, SD-ultra, and SD-ultra+AC10 in wild-type cells. SD-ultra+AC10 is identical to SD-ultra except for the presence of a heteropolymeric (AC)₁₀ spacer 4 nt downstream of the uORF. The sum of the intensities of the bands resulting from cleavage upstream of the uORF was divided by the corresponding sum for cleavage downstream. Data from three biological replicates were used to calculate each mean and standard deviation.

Figure 4.. Meager effect of orthogonal base pairing on downstream cleavage in E. coli.

(A) 5′ UTR of reporter mRNAs containing a stem-loop structure. Arrows, cleavage sites; gray ring, TRAP 11-mer. (B) Relative abundance of 5′ UTR cleavage products for HpGNRA and HpTR8 in wild-type cells. The sum of the intensities of the bands resulting from cleavage upstream of the stem-loop was divided by the corresponding sum for cleavage downstream. Data from three biological replicates were used to calculate each mean and standard deviation. See also Figures S4 and S6.

Figure 5.. Effect of intramolecular coaxial base pairing on distal cleavage in E. coli.

(A) 5′ UTR of reporter mRNAs containing an RNase E cleavage site (GUAUUU) in a hairpin loop (HpHX1) or in an otherwise identical sequence context without the flanking base-paired stem (HX1). Broad black lines, heteropolymeric (AC)_n spacers that were identical in both transcripts. (B) Cleavage within the GUAUUU hexamer of HpHX1 and HX1 in vivo. Equal amounts of total cellular RNA from isogenic wild-type (WT) and ΔrppH (Δ) strains of E. coli containing each reporter mRNA were analyzed by Northern blotting. The bands corresponding to cleavage of the GUAUUU hexamer are indicated. M1 and M2, markers to identify the GUAUUU cleavage products.

Figure 6.. Effects of intermolecular coaxial base pairing on distal cleavage in vitro.

(A) 5′ UTR of RB20 RNA base paired with OLIGO20. Broad black line, 5′ UTR element complementary to OLIGO20. p, 5′ monophosphate; OH, 5′ hydroxyl. (B) Effect of OLIGO20 on the cleavage of monophosphorylated RB20 and AC10 by purified N-RNase E. Monophosphorylated RB20 or AC10 RNA that had been synthesized by in vitro transcription and annealed or not annealed with OLIGO20 bearing a 5′ hydroxyl was treated for 16 min with purified N-RNase E (20 nM). The reaction products were then analyzed by Northern blotting. (C) 5′ UTR of RB20 RNA base paired with OLIGO23. Broad black line, 5′ UTR element complementary to all but the first three nucleotides of OLIGO23. p, 5′ monophosphate; ppp, 5′ triphosphate; OH, 5′ hydroxyl. (D) Effect of OLIGO23 on the cleavage of RB20 by purified N-RNase E. Triphosphorylated (TriP) or monophosphorylated (MonoP) RB20 RNA that had been synthesized by in vitro transcription and annealed with OLIGO23 bearing a 5′ hydroxyl (OH) or monophosphate (MonoP) was treated for 4 min with purified N-RNase E (20 nM). The reaction products were then analyzed by Northern blotting.

Figure 7.. Stabilization of yigL mRNA by coaxial base pairing with SgrS in E. coli.

(A) SgrS-dependent pathway for the degradation of the dicistronic pldB-yigL transcript by RNase E in E. coli. Bent line, SgrS; broad line, SgrS-binding site; black rectangles, pldB and yigL translational units; ppp, 5′ triphosphate; p, 5′ monophosphate. (B) Protection of wild-type yigL mRNA by SgrS in E. coli. Transcription in cells lacking or containing SgrS was arrested by treatment with rifampicin, and equal amounts of total cellular RNA isolated at time intervals thereafter were examined by S1 analysis. (Top) Diagram of the yigL 5′ UTR; (middle) gel images; (bottom) semilogarithmic graph of the amount of yigL mRNA remaining as a function of time. Representative experiments are shown. (C) Rapid degradation of yigLΔE mRNA in E. coli cells lacking SgrS. Dashed line, deleted site of RNase E cleavage and SgrS binding. The graph compares the decay rates of yigLΔE and wild-type yigL in the absence of SgrS. A representative experiment is shown. (D) Inability of SgrS to protect yigL-ST•EM mRNA from rapid degradation in E. coli. ST and EM, complementary RNA segments inserted upstream and downstream of the SgrS binding site. Representative experiments are shown. See also Figure S7. (E) Importance of coaxial base pairing with the yigL 5′ UTR for the ability of SgrS to protect E. coli from α-methylglucoside toxicity. Isogenic E. coli cells whose chromosome contained either a wild-type yigL allele (left) or a yigL-ST•EM allele (right) and in which SgrS was expressed from its native promoter were grown on minimal glycerol medium lacking (top) or containing (bottom) α-methylglucoside (α-MG). To avoid masking the effect of differences in yigL Mrna stability on YigL production and α-methylglucoside resistance, synthesis of the PtsG glucose transporter was rendered insensitive to repression by SgrS in both of these strains.