A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics

Abstract

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.

Keywords: Non-coding rna; l1 stalk; rancRNA; ribosome; translation control.

Figure 1.

rancRNA_18 is a bona fide ncRNA in S. cerevisiae. (A) schematic overview of the truncated versions (Δ1 – Δ9) of the TRM10 locus. the grey bar close to the 5ʹ end depicts the location of rancRNA_18. the presence of rancRNA_18, and the putative 49-mer processing intermediate, was assessed via northern blot analysis (5S rRNA served as loading control). all constructs were expressed from a plasmid in the trm10Δ strain, whereas the original start codon was replaced by a CTG codon to prevent translation of the transcripts (11). ev, empty vector control. (B) growth recovery after 1,050 minutes under hyper-osmotic conditions of wildtype (wt) and trm10Δ cells (black bars) was compared to trm10Δ cells expressing truncated versions of the TRM10 locus. n = 2, mean ± SD. (C) growth of the knock-in strain that expresses the truncated TRM10 construct Δ6 from a novel genomic location (knock-in + ev) in high salt media was compared to the wt cells or to the strain carrying either no additional plasmid (ev) or the plasmid encoding the full-length TRM10 locus (pTRM10). n = 6, mean ± SD. (D), growth competition between a mixture of equal starting amounts of two strains differing only in the absence (white) or presence (grey) of rancRNA_18 expression in normal (no stress) or high salt containing media. cultures were grown to stationary phase and colony forming units (cfu) determined. cultures were then re-diluted and cfu determined at each stationary phase. n = 3, mean ± SD

Figure 2.

Rnt1 is involved in rancRNA_18 biogenesis. (A) secondary structure prediction of the 5 ’end of the TRM10 mRNA with the rancRNA_18 sequence indicated in red and a downstream AGNN tetraloop in orange. (B) growth analysis of a tetracycline (tet)-inducible rnt1 depletion strain (tet_off) in the presence (orange) or absence (green) of doxycycline compared to wildtype (wt, black and blue). n = 6, mean ± SD (C) northern blot analysis on total RNA isolated from wt, trm10Δ, xrn1Δ, rrp6Δ and Rnt1::tet cells either in the absence (-tet) or presence (+tet) of tetracycline. the locations of rancRNA_18 and the putative 49 nucleotides long precursor are indicated. the ethidium-bromide stained tRNAs serve as a loading control. (D) growth of the tetraloop mutant strain (orange) compared to wildtype (black) and trm10Δ (blue) strains was monitored either in normal (left) or high salt media. as a complementation control the trm10Δ strain transformed with a plasmid containing the wt TRM10 locus (green). n = 3, mean ± SD

Figure 3.

rancRNA_18 is a global translational inhibitor. (A) spheroplasts from trm10Δ cells were electroporated with synthetic rancRNA_18 or the mutant version rancRNA-18-M2 and used for metabolic labelling. electroporation in the absence of synthetic RNA (mock) and samples containing the translational inhibitor cycloheximide (CHX) served as controls. radioactively labelled proteins were analysed with SDS-PAGE and autoradiography. coomassie brilliant blue (CBB) stained proteins serve as loading control. signals were quantified and normalized to the no RNA (mock) control. n = 2, mean ± SD. (B) polysome profiling was performed with recovered spheroplasts, RNA was isolated from collected fractions and separated on a denaturing polyacrylamide gel. the distribution of the synthetic rancRNA_18 in the retrieved fractions was visualized by northern blot analysis. (C and D) scatter plots showing translation efficiencies (log2 fold-changes; FC) of sequencing data from total (T) and polysome-associated (P) mRNA from spheroplasts electroporated with no RNA (mock), synthetic rancRNA_18 or rancRNA_18-M2, respectively. different colours indicate mRNAs that show a different association to polysomes in both conditions (green), in samples electroporated with rancRNA_18 only or rancRNA_18-M2 only (blue), in mock samples only (red), or in none of the conditions (grey). p-values for control vs rancRNA_18 data and control vs rancRNA_18-M2 data were <2.2 •10⁻¹⁶. correlation coefficients are indicated as r²

Figure 4.

RancRNA_18 has an influence on A-site tRNA binding. (A) dot blot filter binding analysis was used to analyse binding competition of radiolabeled rancRNA_18 with increasing amounts of LEU2 mRNA to 5 pmol purified yeast ribosomes. the signal in the absence of 80S particles (bkgd) was subtracted. ns, not significant. n = 2; mean ± SD. (B) as in (A) but with increasing amounts of yeast bulk tRNA. The rancRNA_18 binding data (n = 3; mean ± SD; * p < 0.05) are blotted as function of increasing tRNA competitor concentration and the trend-line is depicted. (C) dot blot approach to investigate ribosome binding competition between radiolabeled rancRNA_18 and P-site bound deacylated tRNA^Phe (red). n = 3; mean ± SD. (D) peptidyl transfer between P-site bound N-acetyl-[³H]Phe-tRNA^Phe and A-site located puromycin was assessed in the absence or presence of rancRNA_18. S30 cell extract was used as a control if additional factors are needed for rancRNA_18 functionality. n = 2; mean ± SD. (E) A pre-translocation complex formed on poly(U) mRNA analogues carrying deacylated tRNA^Phe in the P-site (red) and N-acetyl-[³H]Phe-tRNA^Phe in the A-site (green) was used to investigate effects of unlabelled rancRNA_18 addition on tRNA occupancy. extend bound A-site tRNA was assessed via filter binding. n = 6; mean ± SD; * p < 0.05, **** p < 0.0001. (F) as in (E) but in the presence of increasing amounts of rancRNA_18-M2

Figure 5.

Cryo-EM structure of yeast ribosomes with bound rancRNA_18. (A) structure of the 60S ribosomal subunit (wheat) with highlighted rancRNA_18-dependent extra density (red), overlaid with the observed densities for P- and E-site tRNAs (blue) and the L1 stalk (green) in its closed conformation at 4.4 to 5.1 Å resolution. (B) representation of the 60S ribosomal subunit with rRNA in dark grey and ribosomal proteins in light grey. the observed rancRNA_18-dependent extra density (red), the two observed crosslinking site (C2675 and C2693, yellow),) and the L1 stalk (green) in its slight inward position are highlighted. (C) zoom into the rancRNA_18-dependent extra density (red) in close proximity to the observed crosslinking sites (C2675 and C2693, yellow) and r-protein L11 (uL5) (orange)

Figure 6.

RancRNA_18 cross-links to the 60S E-site region. the sites of cross-linking (x-link) of rancRNA_18 carrying two 4-thio-U modifications (4tU-rancRNA_18) to 80S ribosomes were monitored by primer extension analysis and revealed cross-linking sites to C2675 (red) and C2693 (blue) of helix 84 of the 25S rRNA. experiments in the presence of 4tU-rancRNA_18 without irradiation (lane 4) or in the absence of 4tU-rancRNA_18 with irradiation (lane 5) served as controls. cross-linking experiments and primer extension analyses were performed on two biological replicates. the primer extension stop marked with an asterisk could not always be detected using different primers and thus was excluded from further discussions. Lane 1 (ddTTP) and 2 (ddGTP) indicate dideoxy sequencing lanes. location of cross-linking sites (arrows) are shown in the secondary structure model of yeast 25S rRNA (PTC, peptidyl transferase centre)