Dataset Information

Template and target-site recognition by human LINE-1 in retrotransposition.

ABSTRACT: The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease¹. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs². Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

SUBMITTER: Thawani A

PROVIDER: S-EPMC10830416 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Template and target-site recognition by human LINE-1 in retrotransposition.

Thawani Akanksha A Florez Ariza Alfredo Jose AJ Nogales Eva E Collins Kathleen K

Nature 20231214 7997

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease<sup>1</sup>. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs<sup>2</sup>. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed ...[more]

PMID: 38096901

Similar Datasets

Project description:The insertion of mobile elements into the genome represents a new class of genetic markers for the study of human evolution. Long interspersed elements (LINEs) have amplified to a copy number of about 100,000 over the last 100 million years of mammalian evolution and comprise approximately 15% of the human genome. The majority of LINE-1 (L1) elements within the human genome are 5' truncated copies of a few active L1 elements that are capable of retrotransposition. Some of the young L1 elements have inserted into the human genome so recently that populations are polymorphic for the presence of an L1 element at a particular chromosomal location. L1 insertion polymorphisms offer several advantages over other types of polymorphisms for human evolution studies. First, they are typed by rapid, simple, polymerase chain reaction (PCR)-based assays. Second, they are stable polymorphisms that rarely undergo deletion. Third, the presence of an L1 element represents identity by descent, because the probability is negligible that two different young L1 repeats would integrate independently between the exact same two nucleotides. Fourth, the ancestral state of L1 insertion polymorphisms is known to be the absence of the L1 element, which can be used to root plots/trees of population relationships. Here we report the development of a PCR-based display for the direct identification of dimorphic L1 elements from the human genome. We have also developed PCR-based assays for the characterization of six polymorphic L1 elements within the human genome. PCR analysis of human/rodent hybrid cell line DNA samples showed that the polymorphic L1 elements were located on several different chromosomes. Phylogenetic analysis of nonhuman primate DNA samples showed that all of the recently integrated "young" L1 elements were restricted to the human genome and absent from the genomes of nonhuman primates. Analysis of a diverse array of human populations showed that the allele frequencies and level of heterozygosity for each of the L1 elements was variable. Polymorphic L1 elements represent a new source of identical-by-descent variation for the study of human evolution. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF242435-AF242451.]

Dataset Information

Template and target-site recognition by human LINE-1 in retrotransposition.

Publications

Template and target-site recognition by human LINE-1 in retrotransposition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets