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. 2017 Aug 1;8(4):e00730-17.
doi: 10.1128/mBio.00730-17.

RIP-Seq Suggests Translational Regulation by L7Ae in Archaea

Michael Daume  1 , Michael Uhl  2 , Rolf Backofen  2   3 , Lennart Randau  4
Affiliations

RIP-Seq Suggests Translational Regulation by L7Ae in Archaea

Michael Daume et al. mBio. .

Abstract

L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5' untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin, suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria.IMPORTANCE L7Ae is an essential archaeal protein that is known to structure ribosomal RNAs and small RNAs (sRNAs) by binding to their kink-turn motifs. Here, we utilized RIP-Seq methodology to achieve a first global analysis of RNA substrates for L7Ae. Several novel interactions with noncoding RNA molecules (e.g., with the universal signal recognition particle RNA) were discovered. In addition, L7Ae was found to bind to mRNAs, including its own transcript's 5' untranslated region. This feedback-loop control is conserved in most archaea and was incorporated into a reporter system that was utilized to control gene expression in bacteria. These results demonstrate that L7Ae-mediated gene regulation evolved originally in archaeal organisms. The feedback-controlled reporter gene system can easily be adapted for synthetic biology approaches that require strict gene expression control.

Keywords: Archaea; RNA binding proteins; RNA structure; gene regulation.

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Figures

FIG 1
FIG 1
The small RNome and L7Ae-RNA interactome of S. acidocaldarius. (a) Small RNAs (sRNome track) and L7Ae-interacting RNAs (L7Ae sRNA and L7Ae frag RNA tracks) of S. acidocaldarius were subjected to Illumina RNA-Seq. Coverage plots highlight the most abundant RNA reads as distinct peaks. Peaks representing C/D box sRNAs are labeled by (Sac-)sR number in accordance with previous studies (12, 14). Each RNA profile contained one million mapped reads. (b) The coverage plot of the l7ae promoter region of the L7Ae sRNA track is shown. A high number of reads was found downstream of position 1297690, which marks the transcriptional start site (+1) of the 5′ UTR of l7ae. Motifs for the BRE (B recognition element) and TATA sites are boxed upstream of the transcriptional start site. A Shine-Dalgarno (SD) motif is present 9 nt upstream of the GTG start codon, and k-turn-forming Kt-n and Kt-b strands are marked.
FIG 2
FIG 2
L7Ae binds its own 5′ UTR. (a) The schematic structure of the l7ae 5′ UTR k-turn is illustrated. A 3-nt bulge (GAU) is flanked at the 3′ side by two trans sugar-Hoogsteen (arrows) G · A pairs and a 4-bp stem. A single G-C base pair is present at its 5′ side. Mutants produced in this work are marked in red. mut, mutant. (b) EMSA data represent the binding of the l7ae 5′ UTR by recombinant L7Ae. Binding of the native 5′ UTR (Nat RNA) was observed with 10 nM L7Ae and with increasing concentrations (50, 100, and 500 nM L7Ae). The Kt-n strand-mutated 5′ UTR (Kt-n mut1) shows only unspecific binding at a high concentration of 500 nM. (c) Relative levels of β-galactosidase activity (normalized Miller units) are shown for S. acidocaldarius MW001 and Sac-sR10 KO cells that were transformed by the following plasmids: pNat (l7ae promoter plus native 5′ UTR), pKt-n mut1 (l7ae promoter plus Kt-n mutant 1), pKt-n mut2 (l7ae promoter plus Kt-n mutant 2), and pBTmut (BRE/TATA site mutated l7ae promoter plus native 5′ UTR). The assay was performed with strains during logarithmic growth (blue), early stationary growth (red), and late stationary growth (green). The values are normalized to those determined for MW001 plus pNat. Error bars indicate standard deviations of results from five biological replicates. Asterisks (*, Student’s t test; **, Welch’s t test) indicate the significance (P value= <0.05) of the data with respect to the MW001 strain or the Sac-sR10 KO plus pNat strain.
FIG 3
FIG 3
Toxicity effects of L7Ae overproduction in E. coli. The relative levels of GFP signals are shown for E. coli cells that were transformed by different variants of a plasmid which contained a constitutively expressed sfgfp gene and the following IPTG-inducible l7ae or region: the l7ae 5′ UTR upstream of the sfgfp gene, the frameshifted l7ae (no L7Ae), or the control UTR upstream of the sfgfp gene. Error bars indicate standard deviations of results from three biological replicates. Asterisks (*, Student’s t test; **, Welch’s t test) indicate the significance (P value = <0.05) of the data with respect to the strain containing the l7ae 5′ UTR. The GFP fluorescence was recorded from the GFP-positive population only.
FIG 4
FIG 4
L7Ae toxicity can be cured by autoregulation. (a) The growth curves of IPTG-induced E. coli strains containing different variations of the pMD-autol7ae-gfp plasmid are shown as no aKt (5′ UTR of pET plasmid), no L7Ae (frameshifted l7ae), pMD (pMD-autol7ae-gfp), aKt-n mut1, and double aKt-n mut (see Fig. 2a). Three biological replicates were tested. Error bars (standard deviations) are depicted as color-filled areas. (b) The schematic structure of the pMD-autol7ae-gfp plasmid is illustrated. The plasmid contains l7ae under the control of an IPTG-inducible T7 promoter (PT7), which is followed by the l7ae 5′ UTR (yellow streaked box). The presence of the k-turn formed by the 5′ UTR leads to the negative autoregulation of L7Ae translation (autoregulatory k-turn or aKt). The superfolding GFP gene (gfp) is expressed by the constitutive N25 promoter (PN25) from phage T5 (41). The l7ae 5′ UTR (green streaked box) is cloned upstream of the gfp gene and forms a GFP-regulatory k-turn (gfpKt). (c) Data from flow cytometry analysis of E. coli cell populations that carry the pMD-autol7ae-gfp plasmid (pMD) or the aKt-absent variant (no aKt) are illustrated in a dot plot. The pMD strain shows a single population, while the aKt-absent strain displays two cell populations and a larger amount of cell debris. FSC-W, forward-scatter width; FSC-H, forward-scatter height. (d) The relative levels of GFP signals of transformants that comprise mutations in the autoregulatory k-turn are shown. The utilized pMD-autol7ae-gfp plasmids contain a control UTR upstream of the gfp gene to show toxicity-caused GFP downregulation in the aKt mutants. (e) The chart depicts the relative levels of GFP signals of transformants that comprise mutations in the gfp-regulatory k-turn. The values shown in panels d and e were normalized to the pMD strain values. Error bars indicate standard deviations of results from three biological replicates. Asterisks (*, Student’s t test) indicate the significance (P value = <0.05) of the data with respect to the pMD strain.
FIG 5
FIG 5
L7Ae binds the l7ae 5′ UTRs of various archaea. (a) The ~50-bp l7ae upstream sequences of eight taxonomically diverse archaea were extracted from the Clustal Omega alignment (Text S1). The proposed transcriptional start sites (dark gray), Shine-Dalgarno sequences (blue), and start codons (light gray) are marked. Potential Kt-b and Kt-n strands are highlighted in yellow and green, respectively. The GA nucleotides critical for k-turn formation are shown in bold. The sequence of H. volcanii comprises a potential Kt-b strand (highlighted in light blue) in a location apart from a Kt-b strand that was identified further upstream in the alignment (Text S1). The upstream sequence of the P. aerophilum l7ae does not show any of the marked features but comprises a TATA box (red) −25 bp upstream of the start codon. (b) The relative levels of GFP signals of E. coli transformants that comprise a control UTR or the l7ae upstream sequences described for panel a in place of the gfp-regulatory k-turn (except S. acidocaldarius) of the pMD-autol7ae-gfp plasmid are shown. The GFP signals represent the ratio of L7Ae-producing cells to the cells producing no L7Ae (frameshifted l7ae). Error bars indicate standard deviations of results from three biological replicates. Asterisks (*, Student’s t test; **, Welch’s t test) indicate the significance (P value = <0.05) of the data with respect to the control strain. S. aci, Sulfolobus acidocaldarius; A. per, Aeropyrum pernix; S. mar, Staphylothermus marinus; A. ful, Archaeoglobus fulgidus; H. vol, Haloferax volcanii; M. ace, Methanosarcina acetivorans; T. kod, Thermococcus kodakaraensis; M. mar, Methanococcus maripaludis.
FIG 6
FIG 6
K-turn motifs identified in mRNA and SRP RNA are bound by L7Ae. (a) The relative levels of GFP signals of E. coli transformants which contain a control UTR in place of the gfp-regulatory k-turn are depicted. The saci_1468 and saci_2027 strains further contained the respective k-turn mRNA regions identified in the L7Ae RIP-Seq analysis directly downstream of the start codon (GFP fusion) for investigation of the translational regulation of mRNAs by L7Ae (Table S1). The GFP signals represent the ratio of the L7Ae-producing cells to the cells producing no L7Ae (frameshifted l7ae). Error bars indicate standard deviations of results from three biological replicates. Asterisks (**, Welch’s t test) indicate the significance (P value = <0.05) of the data with respect to the control strain. (b) EMSA data demonstrate the binding of L7Ae to the nop5 mRNA Kt. The substrate comprises the first 125 nt of the mRNA, which constitutes the sequence that was found enriched in the L7Ae RIP-Seq analysis (Table S1). Full binding was observed at a concentration of 400 nM L7Ae (L7Ae gradient, 25, 50, 100, 200, 400, and 800 nM). (c) L7Ae shows binding to the k-turn within the SRP RNA of S. acidocaldarius. The substrate consists of the k-turn forming nucleotides 103 to 117 and nucleotides 245 to 259 that were linked by a 4-nt GNAR loop (Text S2). Three secondary structures were formed by the free substrate that showed full binding at an L7Ae concentration of 800 nM (L7Ae gradient, 25, 50, 100, 200, 400, and 800 nM).
FIG 7
FIG 7
Significance of l7ae 5′ UTR binding. (a) The EMSA displays competition analysis of l7ae 5′ UTR (Nat RNA) binding. The Nat RNA is fully bound at a concentration of 400 nM L7Ae (L7Ae gradient, 25, 50, 100, 200, and 400 nM). A 10-fold concentration of unlabeled total RNA of S. acidocaldarius showed effective competition as free Nat RNA was obtained. The total RNA sample was depleted of small RNAs and comprised the 16S and 23S rRNAs as the main molecules (Fig. S1). Almost no competition of the Nat RNA binding was observed for a 100-fold excess of unlabeled C/D box sRNA Sac-sR121. (b) A model highlights the two roles of archaeal L7Ae. L7Ae binds to k-turns in various noncoding RNAs and induces a conformational change which recruits additional proteins. In addition, L7Ae represses the translation of its own mRNA and of other mRNAs by binding to a k-turn structure in the leader or the coding sequence.
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