Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and͞or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3 -azido-3 -deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G 1͞S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G 0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.adenovirus ͉ LINE-1 ͉ quiescent cell ͉ hybrid vector R etrotransposons are mobile elements that insert into new genomic locations by reverse transcription of an RNA intermediate. Human long interspersed element-1 (L1) elements (1, 2) are non-LTR retrotransposons that comprise Ͼ17% of the human genome. The vast majority (Ͼ99%) of L1s are inactive because of point mutations, truncations, and other rearrangements; however, it is estimated that the average human diploid genome contains Ϸ80-100 retrotransposition-competent (RC)-L1s (3). RC-L1s have played and continue to play a significant role in shaping the genome through insertional mutagenesis, nonallelic recombination, and by trans mobilization of non-L1 RNAs (2, 4, 5).L1 retrotransposition requires transcription of L1 RNA, its transport to the cytoplasm, and translation of its two ORFs (ORF1 and ORF2). Both L1-encoded proteins (ORF1p and ORF2p) are thought to preferentially associate with their own encoding RNA (''cis preference'') to form a ribonucleoprotein particle (RNP) (6, 7), which is a proposed retrotransposition intermediate (8,9). The L1 RNP must access the nucleus, where the L1 endonuclease cleaves genomic DNA at a degenerate consensus sequence (5Ј-TTTT͞A and variant sequences) to liberate a 3Ј hydroxyl residue that is subsequently used by the L1 reverse transcriptase (RT) as a primer to copy the L1 sequence in situ, a process termed ''targetprimed reverse transcription'' (2, 10). The resultant L1 cDNA then is joined to target DNA, leading to typical L1 structural hallmarks [5Ј truncations and͞or internal inversions, 3Ј poly (A) tail, and target-site duplications (TSDs)]. Although a putative nucleolar localization signal has been identified in ORF2p (11), it still remains unclear whether the L1 RNP crosses an intact nuclear membrane or whether its entry requires mitotic nuclear envelope breakdown.We previously developed a hybrid vector system consisting of a high-capacity, helper-dependent adenovirus vector encoding a human RC-L1 element (L1.3) tagged with a neomycin-resistance (neo R ) retrotransposition indicator ...

L1 elements are autonomous retrotransposons that can cause hereditary diseases. We have previously identified a full-length L1 insertion in the CHM (choroideremia) gene of a patient with choroideremia, an X-linked progressive eye disease. Because this L1 element, designated L1(CHM), contains two 3'-transductions, we were able to delineate a retrotransposition path in which a precursor L1 on chromosome 10p15 or 18p11 retrotransposed to chromosome 6p21 and subsequently to the CHM gene on chromosome Xq21. A cell culture retrotransposition assay showed that L1(CHM) is one of the most active L1 elements in the human genome. Most importantly, analysis of genomic DNA from the CHM patient's relatives indicated somatic and germ-line mosaicism for the L1 insertion in his mother. These findings provide evidence that L1 retrotransposition can occur very early in human embryonic development.

Despite being scarce in the human genome, active L1 retrotransposons continue to play a significant role in its evolution. Because of their recent expansion, many L1s are not fixed in humans, and, when present, their mobilization potential can vary among individuals. Previously, we showed that the great majority of retrotransposition events in humans are caused by highly active, or hot, L1s. Here, in four populations of diverse geographic origins (160 haploid genomes), we investigated the degree of sequence polymorphism of three hot L1s and the extent of individual variation in mobilization capability of their allelic variants. For each locus, we found one previously uncharacterized allele in every three to five genomes, including some with nonsense and insertion͞deletion mutations. Single or multiple nucleotide substitutions drastically affected the retrotransposition efficiency of some alleles. Onethird of elements were no longer hot, and these so-called cool alleles substantially increased the range of individual susceptibility to retrotransposition events. Adding the activity of the three elements in each individual resulted in a surprising degree of variation in mobilization capability, ranging from 0% to 390% of a reference L1. These data suggest that individual variation in retrotransposition potential makes an important contribution to human genetic diversity.human variation ͉ population genetics ͉ retrotransposon

According to the current model of non-LTR retrotransposon (NLR) mobilization, co-expression of the RNA transposition intermediate, and the proteins it encodes (ORF1p and ORF2p), is a requisite for the formation of cytoplasmic ribonucleoprotein complexes which contain necessary elements to complete a retrotransposition cycle later in the nucleus. To understand these early processes of NLR mobilization, here we analyzed in vivo the protein and RNA expression patterns of the I factor, a model NLR in Drosophila. We show that ORF1p and I factor RNA, specifically produced during transposition, are co-expressed and tightly co-localize with a specific pattern (Loc+) exclusively in the cytoplasm of germ cells permissive for retrotransposition. Using an ORF2 mutated I factor, we show that ORF2p plays no role in the Loc+ patterning. With deletion derivatives of an I factor we define an RNA localization signal required to display the Loc+ pattern. Finally, by complementation experiments we show that ORF1p is necessary for the efficient localization of I factor RNA. Our data suggest that ORF1p is involved in proper folding and stabilization of I factor RNA for efficient targeting, through Loc+ patterning, to the nuclear neighborhood where downstream steps of the retrotransposition process occur.

No abstract

No abstract