Reverse transcriptase and reverse splicing activities encoded by the mobile group II intron cobI1 of fission yeast mitochondrial DNA.
Mobile group II introns encode multidomain proteins with maturase activity involved in splicing and reverse transcriptase (RT) and (often) endonuclease activities involved in intron mobility. These activities are present in a ribonucleoprotein complex that contains the excised intron RNA and the intron-encoded protein. Here, we report biochemical studies of the protein encoded by the group IIA1 intron in the cob gene of fission yeast Schizosaccharomyces pombe mitochondria (cobI1). RNP particle fractions from the wild-type fission yeast strain with cobI1 in its mtDNA have RT activity even without adding an exogenous primer. Characterization of the cDNA products of such reactions showed a strong preference for excised intron RNA as template. Two main regions for initiation of cDNA synthesis were mapped within the intron, one near the DIVa putative high-affinity binding site for the intron-encoded protein and the other near domain VI. Adding exogenous primers complementary to cob exon 2 sequences near the intron/exon boundary stimulated RT activity but mainly for pre-mRNA rather than mRNA templates. Further in vitro experiments demonstrated that cobI1 RNA in RNP particle fractions can reverse splice into double-stranded DNA substrates containing the intron homing site. Target DNA primed reverse transcription was not detected unless a DNA target was used that was already nicked in the antisense strand of exon 2. This study shows that S.pombe cobI1 encodes RNP particles that have most of the biochemical activities needed for it to be a retroelement. Interestingly, it appears to lack an endonuclease activity, suggesting that the active homing exhibited by this intron in crosses may differ somewhat from that of the better-characterized introns.
Schäfer
Bernd
B
Department of Biology IV (Microbiology), Aachen Technical University, Germany. bernd.schaefer@rwth-aachen.de
Gan
Lin
L
Perlman
Philip S
PS
D015152:Blotting, Northern; D015139:Blotting, Southern; D004271:DNA, Fungal / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005091:Exons; D015966:Gene Expression Regulation, Fungal; D007438:Introns / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D018626:Retroelements; D012261:Ribonucleoproteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D012331:RNA, Fungal; D018626:Retroelements; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; C066564:aI2 protein, S cerevisiae; D012194:RNA-Directed DNA Polymerase; D004720:Endonucleases