Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase
- PMID: 8756630
- PMCID: PMC231473
- DOI: 10.1128/MCB.16.9.4726
Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase
Abstract
R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.
Similar articles
-
RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element.Mol Cell Biol. 1995 Jul;15(7):3882-91. doi: 10.1128/MCB.15.7.3882. Mol Cell Biol. 1995. PMID: 7540721 Free PMC article.
-
The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates.J Mol Biol. 2002 Feb 22;316(3):459-73. doi: 10.1006/jmbi.2001.5369. J Mol Biol. 2002. PMID: 11866511
-
DNA-directed DNA polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon.J Mol Biol. 2007 Nov 23;374(2):322-33. doi: 10.1016/j.jmb.2007.09.047. Epub 2007 Sep 20. J Mol Biol. 2007. PMID: 17936300 Free PMC article.
-
Integration, Regulation, and Long-Term Stability of R2 Retrotransposons.Microbiol Spectr. 2015 Apr;3(2):MDNA3-0011-2014. doi: 10.1128/microbiolspec.MDNA3-0011-2014. Microbiol Spectr. 2015. PMID: 26104703 Free PMC article. Review.
-
Evolution of viruses by acquisition of cellular RNA or DNA nucleotide sequences and genes: an introduction.Virus Genes. 2000;21(1-2):7-12. Virus Genes. 2000. PMID: 11022785 Review.
Cited by
-
Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.Nat Biotechnol. 2024 Feb 20. doi: 10.1038/s41587-024-02137-y. Online ahead of print. Nat Biotechnol. 2024. PMID: 38379101
-
Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription.Science. 2023 Apr 21;380(6642):301-308. doi: 10.1126/science.adg7883. Epub 2023 Apr 6. Science. 2023. PMID: 37023171 Free PMC article.
-
Completion of LINE integration involves an open '4-way' branched DNA intermediate.Nucleic Acids Res. 2019 Sep 19;47(16):8708-8719. doi: 10.1093/nar/gkz673. Nucleic Acids Res. 2019. PMID: 31392993 Free PMC article.
-
A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.PLoS One. 2018 Nov 26;13(11):e0207302. doi: 10.1371/journal.pone.0207302. eCollection 2018. PLoS One. 2018. PMID: 30475809 Free PMC article.
-
The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.Genetics. 2017 Jan;205(1):139-153. doi: 10.1534/genetics.116.188680. Epub 2016 Nov 14. Genetics. 2017. PMID: 28049704 Free PMC article.
References
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources