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

Qing Huang; Katarzyna J. Purzycka; Sabrina Lusvarghi; Donghui Li; Stuart F.J. LeGrice; Jef D. Boeke

doi:10.1261/rna.035535.112

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

¹Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
²The High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
³National Cancer Institute, Frederick, Maryland 21702, USA
⁴Laboratory of Structural Chemistry of Nucleic Acids, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznań, Poland
⁵McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA

↵6 These authors contributed equally to this work.

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.