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, 2012, 260909

tRNA-derived Fragments Target the Ribosome and Function as Regulatory Non-Coding RNA in Haloferax Volcanii

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tRNA-derived Fragments Target the Ribosome and Function as Regulatory Non-Coding RNA in Haloferax Volcanii

Jennifer Gebetsberger et al. Archaea.

Abstract

Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5' part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine tuning the rate of protein production.

Figures

Figure 1
Figure 1
Processing and expression of Val-tRF. (a) Secondary structure of H. volcanii Val-tRNA with the Val-tRF depicted in red. Arrowheads indicate the processing positions for the four different observed tRF classes (I–IV). Open arrowheads on the Val-tRNA structure indicate the 3′ ends of the tRFs for class I, as well as the analogous positions for the other tRF classes II, and IV. tRFs from classes I, II, and IV are all processed from the 5′ end of mature tRNAs. Filled arrowheads mark the 5′ and 3′ ends of tRFs derived from class III. Northern blot analyses for Val-tRF were performed using (b) total RNA or (c) ribosome-associated RNA. RNA was isolated from unstressed H. volcanii cells (no stress), or cells grown under different permanent environmental stress conditions (ultraviolet stress (UV), high pH (pH↑), 0.9 M NaCl (NaCl↓), 300 mM MgSO4 (Mg↑)). Arrows indicate the full-length tRNA and the detected processing products. In all panels 5S rRNA served as internal loading control.
Figure 2
Figure 2
tRFs are present in the ribosome-associated RNome. Northern blot analyses of tRFs confirm the presence of class I (a), class II (b), and class III (c) tRFs in the ribosome-associated RNA fraction. The full-length tRNA signals are depicted by black arrows and tRFs by open arrow heads. Approximate lengths of the fragments, as deduced from RNA markers, are indicated. tRFs corresponding to the sequence reads of the cDNA library (Table 1) are underlined. RNA was isolated from unstressed H. volcanii cells (no stress), or cells grown under different permanent environmental stress conditions (ultraviolet stress (UV), high pH (pH↑), 0.9 M NaCl (NaCl↓), 300 mM MgSO4 (Mg↑)). The 5S rRNA (asterisk) of ethidium bromide stained gels served as loading controls.
Figure 3
Figure 3
Val-tRF associates with ribosomes in vitro and in vivo. (a) A representative polysome gradient of H. volcanii. Fractions containing polysomes, 50S, or 30S subunits were collected and used for northern blot analyses. The identity of the individual fractions was confirmed by agarose gel electrophoresis. (b) The presence of the Val-tRF in the different gradient fractions was investigated by northern blot analysis using RNA from unstressed cells or from cultures incubated at high pH (pH 8.5). Arrows indicate the full-length Val-tRNA and the 26 nt long fragment detected in the cDNA library. (c) In vitro filter binding studies of radiolabelled synthetic Val-tRF on ribosomal particles (70S, 50S, 30S) from H. volcanii (left panel). As a positive control, binding of tRNAPhe to E. coli 70S was monitored. To confirm specific binding of Val-tRF an equally long fragment of isoleucine tRNA (Ile-tRF), an RNA sequence not found in our cDNA screen, served as negative control. (Right panel) Quantification of relative binding whereas association of Val-tRF to 70S was normalized to 100%. Signals measured in the absence of any ribosomal particles (-) were subtracted from all experimental points. Error bars show the mean and standard deviation of at least four independent experiments.
Figure 4
Figure 4
Effects of Val-tRF or elevated pH on protein synthesis and cell growth. (a) Val-tRF inhibits H. volcanii in vitro translation. The relative amount of radiolabeled proteins in the absence (-) or in the presence of 3.3 μM Val-tRF is shown. The scrambled version of the Val-tRF (scr) served as specificity control. In all cases, the background values measured in reactions without S30 extracts were subtracted from all experimental points. Error bars represent the mean and standard deviation of at least three independent experiments. (b) Cell growth of unstressed H. volcanii cultures and of cells grown under alkaline conditions (pH 8.5) is shown. Cell density was measured at 600 nm and the average values of two independent growth curves each and their standard deviations are given.
Figure 5
Figure 5
Val-tRF inhibits peptide bond formation. (a) Peptidyl transferase reactions catalyzed by H. volcanii 70S ribosomes (left panel) or 50S subunits (right panel) in the absence (-) or presence of 2.7 μM Val-tRF (a 10-fold molar excess) were performed as described in Material and Methods. The scrambled version of the Val-tRF (scr) as well as Ile-tRF served as specificity controls. The relative amount of the reaction product N-acetyl-[3H]Phe-puromycin (in %) is shown. The product formed in the absence of any tRF (-) was taken as 100%. (b) Val-tRF inhibits peptide bond formation to the same extent regardless if it was added simultaneously with the P-site donor substrate (P) or with the A-site acceptor substrate (A). In all cases, the background values measured in reactions without any ribosomal particles were subtracted from all experimental points. Error bars represent the mean and standard deviation of three to five independent experiments.

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