Exploring Ty1 retrotransposon RNA structure within virus-like particles

Abstract

Ty1, a long terminal repeat retrotransposon of Saccharomyces, is structurally and functionally related to retroviruses. However, a differentiating aspect between these retroelements is the diversity of the replication strategies used by long terminal repeat retrotransposons. To understand the structural organization of cis-acting elements present on Ty1 genomic RNA from the GAG region that control reverse transcription, we applied chemoenzymatic probing to RNA/tRNA complexes assembled in vitro and to the RNA in virus-like particles. By comparing different RNA states, our analyses provide a comprehensive structure of the primer-binding site, a novel pseudoknot adjacent to the primer-binding sites, three regions containing palindromic sequences that may be involved in RNA dimerization or packaging and candidate protein interaction sites. In addition, we determined the impact of a novel form of transposon control based on Ty1 antisense transcripts that associate with virus-like particles. Our results support the idea that antisense RNAs inhibit retrotransposition by targeting Ty1 protein function rather than annealing with the RNA genome.

Figure 1.

Analysis of endogenous reaction products from Ty1 VLPs. (A) Endogenous reverse transcription in CNC− and CNC+ VLPs (VLP amount decreasing from left to right). M: mass markers, and OH: NaOH treatment after reverse transcription (CNC− VLPs, intermediate amount). (B) Products of endogenous reverse transcription in CNC− VLPs in the presence of limiting dNTPs. A, G, T corresponds to the type(s) of dNTP ([α-³²P]-labelled and unlabelled) present in the reaction mixture. (C) Plots of NMIA reactivity of Ty1 ribonucleotides in and around the PBS obtained from CNC− (red) and CNC+ (black) VLPs.

Figure 2.

Structural analysis of the Ty1 PBS region. (A and B) NMIA reactivity plots of ribonucleotides in the PBS region of in vitro transcribed, full-length Ty1 RNA hybridized to tRNA (green), in comparison with that obtained from (A) the same RNA folded in the absence of tRNA (violet), or (B) CNC− VLPs in virio (red). Nucleotide positions corresponding to PBS, Box 0 and Box 1 (13,35) are marked (grey, dashed line). Regions displaying significant changes between each state are noted (I), (II), (III). (C) Model of Ty1 PBS region structure within VLPs. The CYC3 region (15) of the Ty1 genomic RNA is noted (violet).

Figure 3.

RNA secondary structure of the nucleotides 1–1482 of Ty1 genomic RNA (CNC region) in VLPs. This model is based on SHAPE constraints derived from the protein-free state (ex virio) (see ‘Materials and Methods’ section). Colour-coding corresponds to in virio reactivities. Positions (diamonds) within the first 500 nt of Ty1 genomic RNA where reactivity differs in the protein-bound and -free states are marked (blue: increased, grey: decreased). The following regions are annotated: PAL sequences, including reciprocal interstrand interactions (green), and cyclization mediated by CYC5 and CYC3 (violet).

Figure 4.

Effects of protein binding on Ty1 RNA structure. (A) Median reactivities for the CNC region of Ty1 RNA in virio (red) and ex virio (blue) calculated for a rolling window of 155 nt. (B) Analysis of reactivity distributions for +1–500 nt (left) and +501–1482 (right). Boxes outline the middle 50% of each data set with medians (bar) and outliers (circles) shown. (C and D) Reactivity plots of in virio CNC− Ty1 RNA (red) in comparison with (C) ex virio CNC− RNA (blue) and (D) in virio CNC+ (black). Regions showing increased reactivity in ex virio CNC− RNA or in virio CNC+ RNA are boxed.

Figure 5.

Impact of Ty1AS RNA. (A) Reactivity plots of in virio CNC− Ty1 RNA (red) in comparison with in virio CNC+ (black) for the regions encompassing the A-U-G-A-U-G-A heptameric sequence (nucleotides 324–330, 697–703, 1409–1415; dashed rectangles). (B) Ty1 genomic RNA (denoted mRNA) and AS RNA protection. Whole-cell extracts from strains YEM524 (CNC−) and YEM525 (CNC+) were spiked with in vitro transcribed Ty1 sense RNA from the CNC region (∼1.5 kb). Equal aliquots were incubated with (+) or without (−) the nuclease benzonase. RNA extracted from each sample was subjected to northern analysis using ³²P-labelled RNA probes specific for genomic RNA and the added sense RNA or AS RNA I and II. Protection is expressed as a ratio of treated to untreated RNA.