S1 ribosomal protein and the interplay between translation and mRNA decay

Abstract

S1 is an 'atypical' ribosomal protein weakly associated with the 30S subunit that has been implicated in translation, transcription and control of RNA stability. S1 is thought to participate in translation initiation complex formation by assisting 30S positioning in the translation initiation region, but little is known about its role in other RNA transactions. In this work, we have analysed in vivo the effects of different intracellular S1 concentrations, from depletion to overexpression, on translation, decay and intracellular distribution of leadered and leaderless messenger RNAs (mRNAs). We show that the cspE mRNA, like the rpsO transcript, may be cleaved by RNase E at multiple sites, whereas the leaderless cspE transcript may also be degraded via an alternative pathway by an unknown endonuclease. Upon S1 overexpression, RNase E-dependent decay of both cspE and rpsO mRNAs is suppressed and these transcripts are stabilized, whereas cleavage of leaderless cspE mRNA by the unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and that part of the Escherichia coli ribosomes may actually lack S1.

Figure 1.

Ribosomal profile and intracellular distribution of S1 and cspE mRNA. Crude cell extracts were prepared as detailed in ‘Materials and Methods’ section from the following strains and conditions. S1 basal: C-1a exponential culture grown up to OD₆₀₀ = 0.8; S1 overexpression: C-1a/pQE31S1/pREP4 was grown up to OD₆₀₀ = 0.4 and incubated 60 min with 1 mM IPTG to induce rpsA transcription; S1 depletion: C-5699 (araBp-rpsA) grown up to OD₆₀₀ = 0.2 in permissive conditions (LD +arabinose) was diluted 1 : 4 in non-permissive conditions (LD +glucose) to switch off rpsA transcription and further incubated for about 120 min. Cultures were grown at 37°C; before collecting the cells, the cultures were incubated 5 min at 37°C with chloramphenicol (final concentration, 0.1 mg/ml) to prevent polysome dissociation (39). Crude cell extracts (14 OD₂₆₀) were then fractionated by ultracentrifugation on 10–40% sucrose gradients. (A) Ribosomal profile. Thick continuous line: OD₂₆₀ measured for each gradient fractions; grey triangles: S1 distribution. Ten microlitres of the indicated fractions were assayed by western blotting with S1-specific antibodies and the densitometric values (obtained as described in ‘Materials and Methods’ section) were normalized for the highest value obtained. (B) Distribution of S1, PNPase and ribosomal protein L4 in the S100 and 70S fractions. 0.02 OD₂₆₀ of S100 and 70S fractions indicated by arrows in (A) were analysed by western blotting with specific antibodies (as indicated beside the panels). +, S1 basal; ++, S1 overexpression; −, S1 depletion. (C) Intracellular distribution of cspE mRNA. For extracts preparation, 100 ml of culture were taken before and 25 min after addition of 0.4 mg/ml rifampicin and 0.03 mg/ml nalidixic acid (+ rif). RNA was extracted from equal volumes of selected fractions; identical aliquots of RNA samples were loaded on a 6% denaturing polyacrylamide gel and analysed by northern blotting with the CSPE riboprobe. The altered electrophoretic mobility of cspE transcripts observed in the 70S samples (S1 basal, + rif) is probably imputable to the high concentration of ribosomal RNA in those fractions, since these transcripts migrated with the expected mobility upon sample dilution (data not shown).

Figure 2.

Map of plasmid-encoded cspE and rpsO alleles and of endonucleolytic cleavage sites on cspE mRNA. (A) Map of cspE and rpsO alleles cloned in pGM385 plasmid vector. Details about plasmid construction and coordinates of the cloned regions are reported in ‘Materials and Methods’ section. Transcription from cspEp and rpsOp promoters starts at nucleotide 657 473 and 3 309 808, respectively; coordinates of the palindromic region of cspEt and rpsOt transcription terminators are 656 744–656 768 and 3 309 420–3 309 394 (45,69). The promoters, 5′-UTRs and coding regions of the model genes are drawn to scale, whereas 3′-UTRs elements are reported on an arbitrary scale. Dotted line, vector sequence; bent arrow, promoters; hairpin, Rho-independent terminator; black box, HA epitope coding region. The cspE constructs carry the cspEp constitutive promoter. The grey box in pGM397 represents phage λ cI 5′ region. In pGM396 and pGM398, transcription of rpsO::HA alleles is driven by the rpsOp promoter; in pGM398, the 5′-UTR of rpsO was deleted and the transcript from rpsOp produced by the plasmid started with the A of the AUG of the gene (as assessed by primer extension; Supplementary Figure S4). In pGM397, the rpsOp and the 5′-end of the ORF, up to the HA insertion point, were replaced by P_RM and the first 63 codons of λ cI gene, which is naturally leaderless when transcribed from that promoter (70). The black triangles above pGM396 indicate the position of three RNase E cleavage sites mapped in rpsO, M2 (immediately upstream of the rpsOt terminator), M3 (at the beginning of rpsO coding sequence) and M sites (overlapping rpsOt) (69,71,72). (B) Map of endonucleolytic cleavage sites on cspE mRNA. Upward arrows: in vivo cleavage sites on ΔL-cspE; the 5′ ends of degradation products were mapped by primer extension (Figure 3B). Downward arrows: in vitro RNase E-cleavage sites detected on both leadered cspE⁺ and ΔL-cspE (black arrows) or on either cspE⁺ (site 4; data not shown) or ΔL-cspE (site 2) (grey arrows; see Figure 4B). (C) Nucleotide sequence associated with cut sites observed in vivo. The position of the listed sites in cspE mRNA is shown in (B) above. The arrow indicates the cleavage position.

Figure 3.

Analysis of leadered and leaderless cspE alleles transcripts. (A) Expression and stability of leadered and leaderless cspE mRNA upon S1 overexpression. Exponential cultures of C-1a/pREP4/pQE31-S1/pGM924 (cspE⁺) or C-5874/pREP4/pQE31-S1/pGM924 (ΔcspE) ectopically expressing the leaderless cspE allele from pGM924 were grown up to OD₆₀₀ = 0.4 (time = 0) and split in two subcultures. In one subculture (S1 over-expressed) S1 expression was induced by 1 mM IPTG addition and after 60 min (time = 60) the cultures were treated with rifampicin (0.4 mg/ml) and nalidixic acid (0.03 mg/ml). Aliquots for RNA extraction were sampled at times 0 and 60 (no antibiotics) and at different time points after addition of the antibiotics, as indicated (in min) on top of the lanes. Northern blotting was performed as described in ‘Materials and Methods’ section after 6 % denaturing polyacrylamide gel electrophoresis of 5 μg of RNA samples hybridized with radiolabelled CSPE riboprobe (upper panels). (Bottom panels) the gel was stained with ethidium bromide before transfer to check the amount of the loaded RNA samples. The gel portion with 5S rRNA signals is shown. L, leadered cspE chromosomal transcript; ΔL, leaderless cspE plasmid transcript. (B) Primer extension on leaderless cspE RNA degradation products. Selected RNA samples extracted from cultures of C-5868/pGM924 (grown as described in Figure 4A legend) and C-5874/pREP4/pQE31-S1/pGM924 (grown as described here above) were analysed by primer extension with oligonucleotide 2174, as described in ‘Materials and Methods’ section. The positions of different 5′-ends (a, +10; b, +18; c, +31 and d, +54) relatively to the first A of the primary transcript (+1) were defined by running the samples along with DNA sequencing reactions obtained with the same oligonucleotide and plasmid pGM924. The star beside the sequence indicates the A in the AUG initiation codon of cspE gene.1, C-5868/pGM924, 32°C, time 0; 2 and 3, 44°C, 0 and 4 min after rifampicin addition; 4, 5 and 6, C-5874/pREP4/pQE31-S1/pGM924 before (4) and 60 min after (5 and 6) S1 induction by IPTG. Samples 4 and 5 were taken before rifampicin addition (time 0), sample 6, 4 min after. (C) S1 depletion. Bacterial cultures of C-5699/pGM924 were grown up to OD₆₀₀ = 0.4, diluted 1:4 in permissive (+ arabinose, S1 expressed) or non-permissive (+ glucose, S1 repressed) conditions and further incubated until OD₆₀₀ = 0.4–0.5 was reached. RNA was extracted from rifampicin-nalidixic acid treated and untreated samples and northern blotted with radiolabelled 2135 oligonucleotide, as described above (upper panel). (Bottom panel) Ethidium bromide-stained 5S rRNA. L, leadered cspE chromosomal transcript; ΔL, leaderless cspE plasmid transcript. (D) EMSA with purified S1. Radiolabelled cspE⁺ and ΔL-cspE RNAs were synthesized in vitro in the presence of [α³²P]-CTP. The probes (0.7 nM) were incubated 20 min at 21°C without (−) and with increasing amount of S1 (0.1, 0.5, 2.5, 5 and 25 nM). The samples were run on a 5% native polyacrylamide gel.

Figure 4.

Analysis of RNase E role in cspE⁺ and ΔL-cspE degradation. (A) In vivo analysis. Cultures of rne⁺ (C-5869) and rne^ts (C-5868) strains carrying pGM924 were grown to mid-log phase at permissive temperature (32°C) and shifted at non-permissive temperature (44°C). Rifampicin was added immediately before (32°C samples) and 30 min after temperature shift (44°C) and RNA was extracted at the time points indicated on top of the lanes. Five micrograms of RNA samples were analysed by northern blotting with radiolabelled 2135 oligonucleotide. L, leadered cspE chromosomal transcript; ΔL, leaderless cspE plasmid transcript (upper panels). (Bottom panels) ethidium bromide-stained 5S rRNA. (B) In vitro degradation assay. cspE⁺ and ΔL-cspE RNAs (35 nM) 5′-end ³²P-labelled (left) or uniformly radiolabelled with [α³²P]-CTP (right) were incubated at 26°C for the time indicated (in min) above the lanes with 60 ng of purified RNA degradosome and fractionated by 6% PAGE. The size of the main RNA species was estimated by running the samples along with a sequence ladder (data not shown). The corresponding leadered and leaderless RNAs are denoted by the same figure; their respective size (in nucleotides) and 3′-ends (coordinates from NCBI Accession Number U00096.2.) are as follows: 1: 270/229, 656742; 2: 169 (leaderless only), 656 683; 3: 165/127, 656 638. The stars indicate signals that were not reproducibly detected in other experiments. Shorter decay fragments migrating at the bottom of the gel (not shown in the figure), probably corresponding to the probes 3′-end fragments, were present in the right panel.

Figure 5.

Intracellular distribution of leadered and leaderless mRNAs upon modulation of S1 expression. Northern blotting of RNA samples extracted from crude extracts (C) and from ribosomal (R) and S100 (S) fractions. Cultures of C-1a/pREP4/pQE31-S1 (S1 overexpression) or C-5699 (S1 depletion) strains carrying the additional plasmids listed above the panels were grown as detailed in ‘Materials and Methods’ section. Identical volumes of the RNA samples were analysed by 6% denaturing polyacrylamide gel electrophoresis, northern blotted and hybridized with the following oligonucleotides. pGM924, oligo 2135 (cspE panels) or 2469 (specific for rpsO mRNA expressed by the chromosomal gene; rpsOc panel); pGM397, pGM398 (S1 depletion) and pGM929, oligo HA; pGM398 (S1 overexpression), oligo 2313 (specific for rpsO). Left part, −IPTG, S1 basal; + IPTG, S1 induced. Right part, + ARA, S1 expressed, + GLU, S1 depleted.

Figure 6.

Translation of leadered and leaderless mRNAs upon modulation of S1 expression. Proteins extracted from cultures of C-1a/pREP4/pQE31-S1 (S1 depleted; +, S1 basal; ++, S1 induced) or C-5699 (S1 depleted; +, S1 expressed; −, S1 repressed) strains carrying cspE::HA (upper panels: L-cspE, pGM928; ΔL-cspE, pGM929) or rpsO::HA (lower panels: L-rpsO, pGM396; ΔL-rpsO, pGM398; cI-rpsO, pGM397) plasmids were prepared as detailed in ‘Materials and Methods’ section. Proteins were separated by 15% denaturing polyacrylamide gel electrophoresis and immunodecorated with antibodies specific for the HA epitope or, as a loading control, for L4 ribosomal protein. For quantitative evaluation of CspE::HA and RpsO::HA expression, both HA- and L4-specific antibodies signals were quantified with ImageQuant (Molecular Dynamics); each HA-specific signal volume was then normalized by the volume of the corresponding L4-specific signal.