RNA Branching and Debranching in the Yeast Retrovirus-like Element Ty1

Abstract

Ty elements of Saccharomyces cerevisiae are long terminal repeat (LTR) retroelements related to retroviruses. Normal levels of Ty1 transposition require Dbr1p, a cellular enzyme that cleaves 2'-5' RNA bonds. We show that Ty1 RNAs lacking identifiable 5' ends accumulate in virus-like particles (VLPs) in dbr1 mutants. Debranching this RNA in vitro with Dbr1p creates an uncapped version of the normal Ty1 RNA 5' end. We show that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt near the 3' end of the same RNA, creating a lariat. The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.

An evaluation of detection methods for large lariat RNAs

“…Identical electrophoretic behavior was observed for RNA prepared from wildtype samples (data not shown). While we cannot exclude the possibility that single nicks were introduced into lariat Ty1 molecules during RNA preparation, resulting in their migration as a linear species, the mobility of in vivo Ty1 RNA that we observed is consistent with that of previous reports, including the report describing the proposed lariat (Elder et al 1983;Winston et al 1984;Garfinkel et al 1985;Cheng and Menees 2004). Furthermore we are unaware of any previous studies reporting aberrant Ty1 RNA electrophoretic mobility under denaturing conditions.…”

“…2A). One major advantage of using a two-oligonucleotide digestion strategy to detect a lariat RNA is that successful digestion generates a novel RNA species that can be detected on a gel, in contrast to the product of single-oligonucleotide digestion, which Cheng and Menees (2004) report to be indistinguishable from an undigested Ty1 RNA. Furthermore, such a novel branched RNA species would also be observed in RNA samples that had undergone slight degradation or nicking during preparation.…”

“…A recent publication reported evidence suggesting that a very large (> 5000 nucleotides) lariat RNA was an intermediate in Ty1 retrotransposition (Cheng and Menees 2004). The proposed structure of this molecule suggested that it could not possibly have been produced by pre-mRNA splicing because (1) Ty1 contains no known introns and (2) the circular portion of the lariat appeared to have been formed from a linear capped precursor mRNA through some novel mechanism.…”

“…We were unable to detect molecules with such mobilities from in vivo Ty1 RNA preparations. Finally, we evaluated an RNase T1 protection method used by Cheng and Menees (2004) and found this method capable of erroneously reporting the presence of lariat structures when none were present. In short, we were unable to find evidence, using any of these methods, for the proposed lariat structure.…”

“…acrylamide gel electrophoresis (Ruskin et al 1984;Ruskin and Green 1985;Chapman and Boeke 1991;Chen and Sarnow 1995). To test the hypothesis that a lariat Ty1 RNA, similar to that described by Cheng and Menees (2004), would have a lower electrophoretic mobility than that predicted by its molecular weight, we prepared in vitro a large circular Ty1 RNA molecule (corresponding to Ty1 nucleotides 241-5463; all Ty1 coordinates are from Boeke et al 1988) and examined its migration during gel electrophoresis. Such a molecule is very similar in size and overall topology to the proposed lariat and therefore should provide a straightforward and direct assay for the predicted Ty1 lariats.…”