A tRNA-derived fragment competes with mRNA for ribosome binding and regulates translation during stress

Abstract

Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

Keywords: Ribosome; ribosome-associated ncRNA; tRNA-derived fragments; translation regulation.

Figure 1.

Val-tRF inhibits translation in vivo. (A) Under specific stress conditions, particularly under alkaline stress and elevated magnesium conditions (24), the valine tRNA(GAC) isoacceptor is processed to give rise to a 26-residue long 5′-tRF. (B) Metabolic labeling in H. volcanii spheroplasts in the absence (−) or presence of Val-tRF, Ile-tRF or an all DNA version of Val-tRF, named Val-tDF. The antibiotics thiostrepton (Thio) and puromycin (Pmn) served as translation inhibition controls. The background (bkg) represents radioactive bands measured in the absence of any incubation. A representative SDS page of the newly synthesized ³⁵S-labled proteins is shown (upper gel). The coomassie stained gel part underneath the autoradiogram serves as loading control. (C) Quantification of the lane intensities obtained in the metabolic labeling experiments in H. volcanii are shown whereas the activity in the absence of any transformed synthetic RNA (−; mock) is set to 100%. The background values (see above) were subtracted from all other samples. The mean and standard deviations of 4 independent experiments are shown. (D) The H. volcanii Val-tRF also inhibits metabolic labeling in S. cerevisiae spheroplasts. The mean and standard deviations of 4 independent experiments are shown. Cycloheximide (CHX) and an already characterized yeast rancRNA (rancRNA_18) served as inhibition controls. A representative SDS PAGE of newly synthesized proteins after the ³⁵S-methionine spike is shown. The coomassie stained gel part underneath the autoradiogram serves as loading control. In (C) and (D) significant differences relative to the mock control (−) were determined using the 2-tailed paired Student's t-test (***p < 0.001, **p < 0.01, *p < 0.05).

Figure 2.

Val-tRF interferes with the efficient establishment of a translation initiation-like complex as monitored by toeprinting. (A) Schematic representation of the toeprinting assay using H. volcanii 30S subunits (gray) or 70S ribosomes (not shown), initiator tRNA^fMet and an mRNA. Reverse transcription (dotted arrow) of the [³²P]-radiolabeled primer (solid arrow) is terminated in case of a stable 30S/tRNA^fMet/mRNA complex formation. (B) The obtained cDNA products were separated on denaturing polyacrylamide gels and visualized by phosphorimaging. The toeprinting signal depends on the presence of tRNA^fMet and H. volcanii 70S. U, A, C, G denote dideoxy sequencing lanes. The relevant mRNA sequence is shown left to the gel and the start codon (AUG) and the toeprinting sites are highlighted. (C) The toeprinting signals obtained in the presence of H. volcanii 70S ribosomes and tRNA^fMet can be inhibited by increasing amounts of Val-tRF. The Ile-tRF at the highest tested concentration did not interfere with toeprinting and served as specificity control. A representative autoradiograph of a toeprinting gel is shown in the lower panel. Quantification of 4 independent toeprinting experiments is shown above the gel whereas the toeprinting signals in the absence of any added tRF was taken as 100%.

Figure 3.

tRNA and mRNA binding competition with Val-tRF. (A) Radiolabeled Val-tRF was bound to H. volcanii 70S ribosomes in the absence or presence of increasing amounts of unlabeled yeast tRNA. The mean and the standard deviations of 3 independent binding competition experiments are shown underneath the dot blot. Signals measured in the absence of ribosomal particles (−70S) were subtracted from all experimental points. The added molar excess of tRNA over ribosomes is indicated below. (B) Val-tRF binding was monitored as a function of increased mRNA concentration. The mean and the standard deviations of 2 independent binding competition experiments are shown. The added molar excess of mRNA coding for r-protein L12 over ribosomes is shown. The half maximum inhibitory concentration (IC₅₀) is given.

Figure 4.

Val-tRF photo-crosslinking to the 30S ribosomal subunit. (A) The sites of crosslinking at 366 nm of Val-tRF carrying 4-thio uracil, Val-tRF(4SU), to the 970 and 980 regions of the 3′-domain of 16S rRNA of H. volcanii 30S subunits was monitored by primer extension analysis. Experiments in the presence of Val-tRF(4SU) without irradiation or in the presence of unmodified Val-tRF with irradiation served as controls. U, A, C, G denote dideoxy sequencing reactions. The relevant rRNA sequence is shown left to the gel and the crosslinked nucleotides are indicated by asterisks. Note that the reverse transcriptase always stops one nucleotide 3′ of the actual crosslinking site. (B) Sites of Val-tRF(4SU) crosslinking to positions 1403/1404 of 16S rRNA helix 44 in the decoding center were identified by primer extension analysis. U, A, C denote dideoxy sequencing reactions. The relevant rRNA sequence is shown left to the gel and the crosslinked nucleotides are indicated by asterisks. (C) The Val-tRF(4SU)-dependent crosslinks can be competed by the addition of unmodified Val-tRF or mRNA, but not by Ile-tRF. Note that all lanes shown originate from the same gel. Below the autoradiogram image the quantification of the crosslinked bands compare with control bands is given. The intensities of the primer extension stops in the reaction containing Val-tRF(4SU) in the absence of any competitor was taken as 1.0. ‘n.d.’ denotes an experiment not performed in this particular crosslinking experiment shown (refer to Fig. S6 for more data). (D) Val-tRF(4SU) crosslinking in the presence of the antibiotics kanamycin (Kan), neomycin (Neo), spectinomycin (Spt), or tetracycline (Tet).

Figure 5.

Val-tRF crosslinks to sites in close proximity to the 30S mRNA channel. (A) Sites of Val-tRF(4SU) crosslinking in proximity to helix 31 and helix 44 are indicated by red dots in the schematic representation of the E. coli 16S rRNA secondary structure model. (B) Interface view (left) and A-site view (right) of the small ribosomal subunit from T. thermophilus (pdb file 2J00; ⁴⁶). The Val-tRF crosslinking sites are depicted in red and a 30S-bound mRNA piece is shown in green. The 16S rRNA is in blue and the ribosomal proteins are depicted in gray.