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.

A trans -Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

“…However, ectopic expression of individual antisense transcripts does not restore CNC, suggesting that either multiple antisense transcripts or additional factors are required (24,31). Also, nuclease protection and structural probing analyses suggest that although Ty1AS RNAs specifically associate with VLPs from CNC ϩ strains, these transcripts are not packaged into VLPs and do not interact with Ty1 mRNA (32).…”

“…Ty1 CNC is defined by a copy number-dependent decrease in Ty1 retrotransposition and is especially robust in a "Ty1-less" strain of S. paradoxus (28) that may have lost Ty1 elements by LTR-LTR recombination (13). Ty1 CNC acts posttranslationally and in trans, can be overcome by pGTy1 expression, and is characterized by lower levels of mature IN and reverse transcripts (28,31,32). Reduced levels of endogenous Ty1 IN, PR, cDNA, and VLPs are also present in S. cerevisiae (33)(34)(35), which displays CNC (28).…”

Characterizing the functions of Ty1 Gag and the Gag-derived restriction factor p22/p18

“…A major binding site for the CTR and p18 was detected within the pseudoknot 16 present at the 5 0 -end of Ty1 RNA. 19,20 The Ty1 pseudoknot has unusual structural features since it encompasses a large RNA segment (>300 nt). We showed earlier that mutations disrupting pseudoknot formation interfere with retrotransposition, 19 indicating that it provides a critical biological function.…”

“…The pseudoknot structure is absent from in vitro transcribed full-length Ty1 RNA, 20 suggesting that in vivo protein-mediated RNA chaperone activity may be involved in pseudoknot folding process. This hypothesis is in accordance with generally accepted role of nucleic acid chaperone proteins in the RNA folding process.…”

“…16,20 Assuming that Gag and p22/p18 proteins compete for those binding sites and since p22/p18 displays compromised NAC activity, retrotransposition may be targeted at all steps requiring nucleoprotein interactions mediated by Gag (Fig. 2).…”