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. 2022 Sep 10;14(9):2007.
doi: 10.3390/v14092007.

Cell Compartment-Specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome

Małgorzata Zawadzka  1 , Angelika Andrzejewska-Romanowska  1 , Julita Gumna  1 , David J Garfinkel  2 , Katarzyna Pachulska-Wieczorek  1
Affiliations

Cell Compartment-Specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome

Małgorzata Zawadzka et al. Viruses. .

Abstract

The structural transitions RNAs undergo during trafficking are not well understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and those synthesized in vitro, we detected Ty1 gRNA structural alterations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, whereas the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA-RNA interactions required for retrotransposition.

Keywords: Gag; LTR-retrotransposon; RNA genome; RNA structure; Ty1; cell compartment-specific folding; gRNA cyclization; gRNA dimerization; tRNA annealing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Yeast strains used for Ty1 gRNA structural studies and analysis of the ability of NMIA to modify yeast nuclear RNA. (A) Box plot analysis of RT-stop rate measurement with medians for signal and background for U1 snRNA and Ty1 gRNA. Plots present data for approximately 340 nts and 400 nts of U1 snRNA and Ty1 gRNA, respectively. (B) Correlation of position-dependent NMIA and NAI reactivities for U1 snRNA (data for 230 nts). (C) Comparison of DG3408 (pGTy1fs) and DG3412 (pGTy1 wild type) yeast strains.
Figure 2
Figure 2
Analysis of SHAPE reactivities for nuclear Ty1 gRNA compared to cytoplasmic and in vitro full-length Ty1 gRNAs, the inhibition of translation initiation state (for 2500 nts), and the in virio state (for 1482 nts). (A) Violin plot analysis of SHAPE reactivity distributions with medians. (B) Pie chart analysis of SHAPE reactivity percentage distribution. (C) Correlation of position-dependent NMIA reactivities. (D) The median SHAPE reactivity profiles smoothed within a 75-nt window. (E) The median profile of absolute SHAPE reactivity change between nuclear and cytoplasmic datasets, smoothed within a 75-nt window. Light-red shadings indicate regions with the greatest absolute differences above the global median change (marked in red) (see Section 2).
Figure 3
Figure 3
SHAPE-directed structure model of the nuclear Ty1 gRNA and its comparison with MFE structures predicted for cytoplasmic or in vitro Ty1 gRNAs. (A) Genomic organization of Ty1 gRNA. (B) Box plot analysis of SHAPE reactivities mapped to single- and double-stranded regions of the nuclear Ty1 gRNA MFE structure. (C) Pie chart analysis of percentage distributions of nucleotide pairings in the MFE structures, including high probability base pairs (HP bps, pairing probability > 80%). (D) Box plot analysis of MFE bps and (E) HP bps distance with medians. (F) Venn diagram showing overlap of HP bps. (G) Sensitivity and PPV parameters for lowSS regions identified in nuclear Ty1 gRNA. (H) LowSS regions analysis. Light shadings indicate extending to encompass entire intersecting helices from MFE structures. The bottom bars present locations of lowSS regions in the cytoplasmic Ty1 gRNA. Overlapped regions are marked by *. Significance was computed by unpaired two-tailed Mann–Whitney test; **** p < 0.0001. * p < 0.05; n.s., not significant.
Figure 4
Figure 4
Comparative analysis of Ty1 gRNA regions containing cis-acting sequences. (A) The step plot (top) and the difference plot (down) calculated by subtracting the in virio NMIA reactivities from those of the nuclear Ty1 gRNA. (B) The MFE models of nuclear and cytoplasmic Ty1 gRNA 5′ end. Cis-acting sequences are marked with colored boxes. Neighboring regions affected by tRNAiMet annealing are boxed.
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