Cited by 520Chromosomal and Genetic VariationsGenomic variations and chromosomal abnormalitiesGenomics and Chromatin Dynamics

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Human L1 element target‐primed reverse transcription in vitro

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Cost, Gregory J., Feng, Qinghua, Jacquier, Alain, Boeke, Jef D.·2002·The EMBO Journal

DOI 10.1093/emboj/cdf592PMC PMC131089

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Abstract

L1 elements are ubiquitous human transposons that replicate via an RNA intermediate. We have reconstituted the initial stages of L1 element transposition in vitro. The reaction requires only the L1 ORF2 protein, L1 3' RNA, a target DNA and appropriate buffer components. We detect branched molecules consisting of junctions between transposon 3' end cDNA and the target DNA, resulting from priming at a nick in the target DNA. 5' junctions of transposon cDNA and target DNA are also observed. The nicking and reverse transcription steps in the reaction can be uncoupled, as priming at pre-existing nicks and even double-strand breaks can occur. We find evidence for specific positioning of the L1 RNA with the ORF2 protein, probably mediated in part by the polyadenosine portion of L1 RNA. Polyguanosine, similar to a conserved region of the L1 3' UTR, potently inhibits L1 endonuclease (L1 EN) activity. L1 EN activity is also repressed in the context of the full-length ORF2 protein, but it and a second cryptic nuclease activity are released by ORF2p proteolysis. Additionally, heterologous RNA species such as Alu element RNA and L1 transcripts with 3' extensions are substrates for the reaction.

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Authors: Cost, G. J., Feng, Q., Jacquier, A., & Boeke, J. D.
Affiliations: Centre National de la Recherche Scientifique (FR)
Johns Hopkins University (US)
Corresponding author: Boeke, Jef D.
ORCID: Cost, Gregory J.
Feng, Qinghua
Jacquier, Alain
Boeke, Jef D.
Venue: The EMBO Journal
Publisher: Springer Science and Business Media LLC
Published: 2002-11-01
Volume: 21
Issue: 21
Pages: 5899-5910
ISSN: 0261-4189 (ISSN-L)
1460-2075
DOI: 10.1093/emboj/cdf592
PMC: PMC131089
OpenAlex: W1965141089
Cited by: 519
References: 51
Citations by year: 2026: 18 · 2025: 19 · 2024: 17 · 2023: 23 · 2022: 24 · 2021: 28 · 2020: 25 · 2019: 25 · 2018: 21 · 2017: 34 · 2016: 19 · 2015: 25 · 2014: 20 · 2013: 33 · 2012: 24
FWCI: 21.05
Type: journal article
Language: en
Open access: bronze
doi.org
Licence: www.springer.com/tdm (tdm)
www.springer.com/tdm (vor)
Locations: The EMBO Journal (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health
National Cancer Institute (CA-16519, P01CA016519)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Chromosomal and Genetic Variations
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords: target-primed reverse transcription, ORF2 protein, DNA nicking
MeSH terms: Base Sequence, DNA Transposable Elements, DNA Replication, Humans, Molecular Sequence Data, RNA, Transcription, Genetic, DNA, Complementary

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Human L1 element target‐primed reverse transcription in vitro

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