Relationship between RNA lariat debranching and Ty1 element retrotransposition.
The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants 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 of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in 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 to ACT1 intron RNA. Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant 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 of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.
Salem
Laura A
LA
School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Boucher
Christopher L
CL
Menees
Thomas M
TM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme