Non-cognate template usage and alternative priming by a group II intron-encoded reverse transcriptase.
Group II introns are retroelements that site-specifically insert into DNA through homing. They are also implicated in related phenomena such as ectopic site insertions and precise intron deletions, but little is known about how group II intron reverse transcriptases (RTs) interact with non-cognate substrates. Here we show that wild-type aI2 RT readily reverse transcribes non-cognate RNAs in mitochondrial RNP particles when the aI2 intron structure is misfolded. In two closely related priming mutants, 1degree2(DeltaD5) and 1(+)2(DeltaD5), which contain wild-type RT but a disrupted intron structure, the RT has substantially lost specificity for aI2 RNA and copies multiple RNAs present in the RNP particles, using an alternative priming mechanism. The RT in 1degree2(DeltaD5) RNP particles can also copy exogenous RNAs but unlike the endogenous templates, a complementary primer is required, suggesting that the alternative priming event is specific to RT-RNA interactions formed in vivo. Alternatively primed cDNAs from strains 1degree2(DeltaD5), 1(+)2(DeltaD5) and 1degree2(P714T) (containing the mutation P714T in the RT) do not use homing site DNA as a primer, but appear to utilize a non-complementary DNA primer of approximately ten nucleotides. The alternative priming mechanism and reverse transcription of non-cognate templates has implications for in vivo reverse transcription of non-intronic RNAs, which is expected to occur during intron deletions and other retroprocessing events.
Morozova
Tatiana
T
Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
Seo
Wooseok
W
Zimmerly
Steven
S
D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics*; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics; D023282:Nuclease Protection Assays; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D018626:Retroelements / Q000235:genetics*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D024363:Transcription Initiation Site; D015003:Yeasts / Q000166:cytology / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D018626:Retroelements; D012261:Ribonucleoproteins; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D015719:Single-Strand Specific DNA and RNA Endonucleases