Inefficient and incorrect processing of the Ac transposase transcript in iae1 and wild-type Arabidopsis thaliana.
As part of the analysis of the Arabidopsis mutant iae1-1 (increased Ac excision), quantitative studies of the Ac transposase transcript were conducted. The primary transcript of Ac contains three small introns (introns 1-3; mean size 89 bp) and one larger intron (intron 4; 387 bp). We analysed the splicing of intron 3 and intron 4 in wild-type Arabidopsis and the iae1-1 mutant. Our results demonstrate that the splicing of Ac introns 3 and 4 is inefficient (splicing efficiencies 57 and 30% respectively) compared with that of an intron of an endogenous Arabidopsis gene (PHYB intron 1; splicing efficiency 90%). The poor splicing efficiency of Ac intron 4 was found to correlate with aberrant processing. Steady state levels of total Ac transcript were higher in the iae1-1 mutant than wild-type, but the same aberrant processing occurred. The inefficient processing of Ac in Arabidopsis prompted us to construct an Ac element lacking introns (Ac::cDNA) in an attempt to increase transposition frequencies. Autonomous activity of the Ac::cDNA element was undetectable in Arabidopsis, despite its ability to transpose at high frequency in response to a strong transposase source (35S::transposase) in trans, and the demonstrable autonomy of the same element in tobacco. A number of smaller transcripts were detected in Arabidopsis lines containing Ac::cDNA or Ac. Analysis of these smaller transcripts revealed a high frequency of premature polyadenylation in exon 2 and splicing of cryptic introns.
Jarvis
P
P
Department of Molecular Genetics, John Innes Centre, Norwich Research Park, Colney, UK.
Belzile
F
F
Dean
C
C
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D046508:Culture Techniques; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D018506:Gene Expression Regulation, Plant; D007438:Introns; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D018749:RNA, Plant / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014026:Nicotiana / Q000235:genetics; D019895:Transposases
D012322:RNA Precursors; D018749:RNA, Plant; D004254:DNA Nucleotidyltransferases; D019895:Transposases