Characterization of LINE-1 Ribonucleoprotein Particles

Stanford University, United States of America
PLoS Genetics (Impact Factor: 7.53). 10/2010; 6(10). DOI: 10.1371/journal.pgen.1001150
Source: PubMed


Author Summary
Long Interspersed Element-1 (LINE-1 or L1) sequences are the predominant class of autonomous retrotransposons in the human genome and comprise an astounding 17% of human DNA. Although the majority of L1s are considered to be “dead,” an average human genome contains ∼80–100 active L1s. Active L1s encode two proteins (ORF1p and ORF2p) that are required for mobility (retrotransposition) by a “copy and paste” mechanism termed target-site primed reverse transcription. Prior experiments suggested that ORF1p, ORF2p reverse transcriptase activity, and L1 mRNA associate in ribonucleoprotein (RNP) particles and that RNP formation is a necessary step in L1 retrotransposition. However, the difficulty in detecting ORF2p from engineered human L1s has prevented a thorough understanding of its role in L1 retrotransposition. Here, we have exploited epitope and/or RNA–tagging strategies to detect and characterize a “basal” RNP complex from engineered human L1s. We also expanded on previous studies and characterized how mutations in conserved functional domains of ORF1p and ORF2p can adversely affect L1 RNP formation/function. Finally, our strategy allowed us to detect the L1–encoded proteins and L1 RNA in cytoplasmic foci. Thus, we have developed and employed a system to gain greater understanding of LINE-1 retrotransposition at the molecular level.

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    • "By contrast, the role of the ORF2 protein is best known, having its 146-kDa multifunctional polypeptide simultaneously endonuclease and reverse transcriptase activities, crucial for retrotransposition (e.g. Dewannieux and Heidmann 2005; Doucet et al. 2010), although presenting a cysteine-rich domain with a still unknown function (Fanning and Singer 1987; Dai et al. 2011). LINE-1 sequences in the mammalian genomes have traditionally been referred as selfish elements, persisting over time due to their replicative advantage above the host genome. "
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    ABSTRACT: Long interspersed nuclear elements-1 (LINE-1) are the most abundant and active retrotransposons in the mammalian genomes. Traditionally, the occurrence of LINE-1 sequences in the genome of mammals has been explained by the selfish DNA hypothesis. Nevertheless, recently, it has also been argued that these sequences could play important roles in these genomes, as in the regulation of gene expression, genome modelling and X-chromosome inactivation. The non-random chromosomal distribution is a striking feature of these retroelements that somehow reflects its functionality. In the present study, we have isolated and analysed a fraction of the open reading frame 2 (ORF2) LINE-1 sequence from three rodent species, Cricetus cricetus, Peromyscus eremicus and Praomys tullbergi. Physical mapping of the isolated sequences revealed an interspersed longitudinal AT pattern of distribution along all the chromosomes of the complement in the three genomes. A detailed analysis shows that these sequences are preferentially located in the euchromatic regions, although some signals could be detected in the heterochromatin. In addition, a coincidence between the location of imprinted gene regions (as Xist and Tsix gene regions) and the LINE-1 retroelements was also observed. According to these results, we propose an involvement of LINE-1 sequences in different genomic events as gene imprinting, X-chromosome inactivation and evolution of repetitive sequences located at the heterochromatic regions (e.g. satellite DNA sequences) of the rodents' genomes analysed.
    Journal of applied genetics 08/2014; 56(1). DOI:10.1007/s13353-014-0241-x · 1.48 Impact Factor
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    • "This mRNA is transported to the cytoplasm where ORF1p and ORF2p are translated [27] [28]. Notably, the L1-encoded proteins tend to associate with the L1 mRNA used during translation (a process called cis-preference [29]) and are thought to form a ribonucleoprotein particle (RNP) that is a proposed retrotransposition intermediate [30] [31] [32] [33]. Once in the nucleus, L1 integration occurs by a mechanism termed target primed reverse transcription (TPRT) [34] [35]. "
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    ABSTRACT: Transposable Elements are pieces of DNA able to mobilize from one location to another within genomes. Although they constitute more than 50% of the human genome, they have been classified as selfish DNA, with the only mission to spread within genomes and generate more copies of themselves that will ensure their presence over generations. Despite their remarkable prevalence, only a minor group of transposable elements remain active in the human genome and can sporadically be associated with the generation of a genetic disorder due to their ongoing mobility. Most of the transposable elements identified in the human genome corresponded to fixed insertions that no longer move in genomes. As selfish DNA, transposable element insertions accumulate in cell types where genetic information can be passed to the next generation. Indeed, work from different laboratories has demonstrated that the main heritable load of TE accumulation in humans occurs during early embryogenesis. Thus, active transposable elements have a clear impact on our pluripotent genome. However, recent findings suggest that the main proportion of fixed non-mobile transposable elements might also have emerging roles in cellular plasticity. In this concise review, we provide an overview of the impact of currently active transposable elements in our pluripotent genome and further discuss new roles of transposable elements (active or not) in regulating pluripotency. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 07/2014; 1849(4). DOI:10.1016/j.bbagrm.2014.07.007 · 6.33 Impact Factor
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    • "We reasoned that if A3A specifically inhibits L1 EN activity, ENi L1 retrotransposition events should escape A3A inhibition. To test this hypothesis, we co-transfected a human L1 (either WT pAD2TE1 or EN-deficient pAD136 [Doucet et al., 2010]) and a WT A3A expression construct into Chinese Hamster Ovary (CHO) cell lines that are NHEJ-proficient (4364a) or NHEJdeficient (XR-1) (Morrish et al., 2002, 2007). A3A expression inhibited WT L1 retrotransposition in both cell lines, and also inhibited ENi retrotransposition in XR-1 cells (Figure 1C). "
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    ABSTRACT: Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition. DOI:
    eLife Sciences 04/2014; 3(3):e02008. DOI:10.7554/eLife.02008.001 · 9.32 Impact Factor
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