Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

dc.contributor.author	Penno, Christophe
dc.contributor.author	Kumari, Romika
dc.contributor.author	Baranov, Pavel V.
dc.contributor.author	van Sinderen, Douwe
dc.contributor.author	Atkins, John F.
dc.contributor.funder	Science Foundation Ireland
dc.contributor.funder	Irish Research Council
dc.date.accessioned	2017-10-18T09:40:13Z
dc.date.available	2017-10-18T09:40:13Z
dc.date.issued	2017
dc.description.abstract	RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3′ of such ‘slippage-stimulatory’ structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem–loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5′ adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited.	en
dc.description.status	Peer reviewed	en
dc.description.version	Published Version	en
dc.format.mimetype	application/pdf	en
dc.identifier.citation	Penno, C., Kumari, R., Baranov, P. V., van Sinderen, D. and Atkins, J. F. (2017) 'Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage', Nucleic Acids Research, 45(17), pp. 10143-10155. doi: 10.1093/nar/gkx689	en
dc.identifier.doi	10.1093/nar/gkx689
dc.identifier.endpage	10155
dc.identifier.issn	0305-1048
dc.identifier.issn	1362-4962
dc.identifier.issued	17
dc.identifier.journaltitle	Nucleic Acids Research	en
dc.identifier.startpage	10143
dc.identifier.uri	https://hdl.handle.net/10468/4888
dc.identifier.volume	45
dc.language.iso	en	en
dc.publisher	Oxford University Press	en
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Research Centres/12/RC/2273/IE/Alimentary Pharmabiotic Centre (APC) - Interfacing Food & Medicine/
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Investigator Programme/12/IA/1335/IE/Development of computational resources for the analysis of Genome Wide Information on Protein Synthesis (GWIPS)./
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Investigator Programme/13/IA/1853/IE/Dynamic redefinition of codons: From antivirals to an essential micronutrient/
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Investigator Programme/12/IP/1492/IE/Using ribosome profiling to study translation initiation/elongation and facilitate optimization of protein synthesis/
dc.relation.uri	https://academic.oup.com/nar/article/45/17/10143/4064206/Stimulation-of-reverse-transcriptase-generated
dc.rights	© 2017, the Authors. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.	en
dc.rights.uri	https://creativecommons.org/licenses/by-nc/4.0/
dc.subject	DNA-directed RNA polymerase	en
dc.subject	Retroviridae	en
dc.subject	RNA-directed DNA polymerase	en
dc.subject	Enzymes	en
dc.subject	RNA	en
dc.title	Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage	en
dc.type	Article (peer-reviewed)	en