tRNA-derived short RNAs bind to Saccharomyces cerevisiae ribosomes in a stress-dependent manner and inhibit protein synthesis in vitro

Abstract

Recently, a number of ribosome-associated non-coding RNAs (rancRNAs) have been discovered in all three domains of life. In our previous studies, we have described several types of rancRNAs in Saccharomyces cerevisiae, derived from many cellular RNAs, including mRNAs, rRNAs, tRNAs and snoRNAs. Here, we present the evidence that the tRNA fragments from simple eukaryotic organism S. cerevisiae directly bind to the ribosomes. Interestingly, rancRNA-tRFs in yeast are derived from both, 5'- and 3'-part of the tRNAs and both types of tRFs associate with the ribosomes in vitro The location of tRFs within the ribosomes is distinct from classical A- and P-tRNA binding sites. Moreover, 3'-tRFs bind to the distinct site than 5'-tRFs. These interactions are stress dependent and as a consequence, provoke regulation of protein biosynthesis. We observe strong correlation between tRF binding to the ribosomes and inhibition of protein biosynthesis in particular environmental conditions. These results implicate the existence of an ancient and conserved mechanism of translation regulation with the involvement of ribosome-associating tRNA-derived fragments.

Keywords: Saccharomyces cerevisiae; protein biosynthesis regulation; rancRNAs; ribosome; tRFs; tRNA-derived small RNAs.

Figure 1.

Secondary structures of S. cerevisiae tRNAs with marked positions of tRFs used in this study. Secondary structures His-tRNA (GTG), Ser-tRNA (AGA), Gly-tRNA (GCC), Leu-tRNA (TAA) and Thr-RNA (TGT) are presented. tRFs sequence is depicted in colors.

Figure 2.

tRFs associate with S. cerevisiae ribosomes. Saturation assays of radiolabeled synthetic tRFs binding to 80S ribosome. tRF/ribosome ratio is presented (1:10, 1:5, 1:2, 1:1, 2:1, 5:1 and 10:1). Binding efficiency is shown in [%].

Figure 3.

Yeast 3′-tRFs occupy the same ribosomal binding site which is distinct from the classical A site, P site and 5′-tRF-His (GTG) binding site. (A and B) Dot-blot representatives and (C and D) graphical representation of in vitro competition binding studies on the ribosomes with 3′-tRF-Leu (TAA) pre-bound (A and C) or with 5′-tRF-His (GTG) pre-bound (B and D). 5–50 pmol of corresponding competitor were used, marked with color boxes on (A and B) and color lines on (C and D). Dot blots in the black boxes correspond to the input 3′-tRF-Leu (TAA) (A) or input 5′-tRF-His (GTG) (B).

Figure 4.

tRFs associate with S. cerevisiae ribosomes in a stress-dependent manner. tRF/ribosome in vitro binding efficiency is shown as percentage of input tRF used for the binding reaction. Individual stress conditions used for ribosome isolation are marked on both A and B with corresponding colors.

Figure 5.

tRFs inhibit polypeptide synthesis in S. cerevisiae. Translational efficiency is shown as percentage of the activity of control experiment without tRFs. (A) Dose-dependent tRF effect on yeast poly(Phe) synthesis in the isolated in vitro translation system. (B) Stress-dependence of tRFs inhibitory effect on yeast poly(Phe) synthesis in the isolated in vitro translation system.

Figure 6.

Correlation between tRF association with the ribosomes and tRF influence on translation. Values are means of replicates which are fully presented on Figs 5 and 6. Ribosomes were isolated from S. cerevisiae grown under 12 different environmental conditions.