Retrotransposon Ty1 RNA contains a 5′-terminal long-range pseudoknot required for efficient reverse transcription

Abstract

Ty1 retrotransposon RNA has the potential to fold into a variety of distinct structures, mutation of which affects retrotransposition frequencies. We show here that one potential functional structure is located at the 5 ′ end of the genome and can assume a pseudoknot conformation. Chemoenzymatic probing of wild-type and mutant mini-Ty1 RNAs supports the existence of such a structure, while molecular genetic analyses show that mutations disrupting pseudoknot formation interfere with retrotransposition, indicating that it provides a critical biological function. These defects are enhanced at higher temperatures. When these mutants are combined with compensatory changes, retrotransposition is restored, consistent with pseudoknot architecture. Analyses of mutants suggest a defect in Ty1 reverse transcription. Collectively, our data allow modeling of a three-dimensional structure for this novel critical cis-acting signal of the Ty1 genome.

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme

“…Information about pairing patterns within the pseudoknots was introduced into the RNAComposer dot-bracket annotated input using square brackets. The structure of each potential pseudoknot was additionally refined using distance restraints derived from canonical A-RNA structure (Supplemental Table S9) applying the described procedure (Huang et al 2013). In some cases, the generated 3D structures did not correlate with long-distance interactions or relative positioning of the helixes deduced from the provided experimental data (i.e., Puzzle 8).…”

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

“…However, the 2-fold decrease in cDNA accumulation cannot explain the 50-fold decrease in retrotransposition frequency. 19 Discovery of the p22/ p18 restriction factor sheds new light on retrotransposition regulation, and in fact, may explain results obtained from mutational analysis of the pseudoknot. The fact that p18 binds Ty1 RNA in vitro within pseudoknot raises the possibility that mobility defects observed for RNA pseudoknot mutants might relate to interactions with 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.…”

“…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. 21 We found more than 50-fold decrease in transposition when the pseudoknot was destabilized in UC264AG mutant.…”

“…21 We found more than 50-fold decrease in transposition when the pseudoknot was destabilized in UC264AG mutant. 19 Analysis of the Ty1 RNA half-life, packaging of Ty1 RNAs into VLPs and cDNA accumulation suggest that the primary importance of the pseudoknot occurs during reverse transcription. However, the 2-fold decrease in cDNA accumulation cannot explain the 50-fold decrease in retrotransposition frequency.…”