Selection of RNA aptamers imported into yeast and human mitochondria

Abstract

In the yeast Saccharomyces cerevisiae, nuclear DNA-encoded is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA(Lys) and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA(Lys) in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 3'-end and the TPsiC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.

FIGURE 1.

(A) Cloverleaf structures of yeast lysine tRNAs, partially imported into mitochondria tRK1 and nonimported tRK2. The import determinants identified previously are indicated by arrows on the tRK2 structure. The tRK1 regions determined by oligonucleotides used for RT-PCR are shown by bold lines, residues that have been randomized for SELEX experiment are circled, and regions conserved in all the selected RNA aptamers are indicated by thin lines. Selected substitutions in the D-arm of tRK1 are shown by arrows. (B,C) Strategy for in organello RNA selection. Scheme of randomized RNA library selection (B) for import into isolated yeast mitochondria and (C) for both affinity to preMSK1p and mitochondrial import. (D,E) Characterization of RNA pools resulting from different rounds of selection: (D) preMSK1p-binding capacity; (E) efficiency of mitochondrial import, in percent relative to tRK1. (S0) Initial RNA library; (SI2–SI8) RNA pools after two to eight rounds of selection for the import into mitochondria; (SIM2–SIM7) RNA pools after two to seven rounds of selection for preMSK1p binding and mitochondrial import. RNAs marked by * were aminoacylated prior to the test. All experiments were performed at least twice in an independent way; standard deviations values are indicated.

FIGURE 2.

Secondary structure predictions and in-gel FRET analysis of selected RNA aptamers. (A,B) Secondary structure prediction for RNA aptamers of (A) SI and (B) SIM series. Two types of structure are shown for each RNA, with a canonical aminoacceptor (AA) stem and with an alternative F-stem. (Red) Nucleotides corresponding to the tRK1 AA-stem; (blue) to the D-arm; (purple) to the TΨC stem–loop; (black) stably selected substitutions and variable regions; fluorophore positions are shown by splashes; (Fl) fluorescein. (C) In gel FRET analysis of two selected RNA aptamers, (left panel) SI 56 and (right panel) SIM 92. Radioactive signals, p32, and fluorescence (ex. 488 nm, em. 520 nm), Fl, of increasing amounts of RNAs labeled by donor fluorophore only (Fl-RNA) or by both donor and acceptor fluorophores (FlDy-RNA), in a native 12% PAGE.

FIGURE 3.

Study of conformational rearrangements in the tRK1 molecule by the FRET approach. (A) Secondary structures of tRK1 predicted by Mfold. tRK1 domains are colored as in Figure 2, and fluorophore positions are shown by splashes. Post-transcriptional modifications are indicated only in the tRK1 regions involved in the alternative structure. (B–D) In-gel FRET analysis of (B) tRK1, (C) tRK1 complex with preMSK1p, and (D) tRK1 complex with Eno2p. Radioactive signals (p32) and fluorescence (Fl) in native 9% PAGE are presented, as in Figure 2D.

FIGURE 4.

Structure and mitochondrial import of small synthetic RNAs. (A) Short RNA molecules based on the sequence of F1 (25-nt fragment of the tRK1 3′-part). Nucleotide substitutions are colored. (B) Examples of in vitro import test for the labeled short RNAs and its import efficiencies; see also Table 4. (C) Structure of synthetic RNA molecules composed of joined F-helix, D-arm, and/or aminoacceptor (AA-) stem of tRK1/tRK2. For several versions two alternative structures are shown. AA-stem regions are shown in red for tRK1 and in orange for tRK2; D-arms are in blue for tRK1 and in green for tRK2. (D) Dependence of synthetic RNA import efficiencies on the F-stem free energy (Mfold predicted).

FIGURE 5.

Import of small synthetic RNAs into human mitochondria. (A) Example of ³²P-labeled RNAs import into isolated HepG2 mitochondria in the presence of human import-directing proteins. The mitochondrial import efficiencies were calculated as the average of three independent experiments; standard deviation values are indicated in Table 4. (B) Northern blot analysis of presence of tRK1 (T7 transcript) and synthetic RNA F1D1 in total and mitochondrial RNA preparations 48 h after transient transfection of HepG2 cells. Probes used for hybridization are indicated in the left column. Import efficiency of RNAs was estimated as a ratio between the signal obtained after hybridization with the anti-tRK1 probe and that obtained after hybridization with an oligonucleotide against the mitochondrial tRNA^Thr in the mitochondrial RNA preparation. In order to compare import efficiencies of different RNAs, the values obtained were divided by the ratios calculated in the same way but in a total RNA preparation, which indicate the total level of each RNA in the transfected cells. The resulting normalized import efficiency of the tRK1 transcript was taken as 1.