Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition

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Abstract
TheSaccharomyces cerevisiae DBR1gene encodes a 2′-5′ phosphodiesterase that debranches intron RNA lariats following splicing. Yeastdbr1mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection ofdbr1mutant alleles to explore the relationship between the roles ofDBR1in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize toACT1intron RNA. Protection patterns confirm that sequences from the 5′ end of the intron to the lariat branch point accumulate indbr1mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generateddbr1mutant alleles are also deficient for debranching, further supporting a role for 2′-5′ phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently ofDBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.