Preferential insertion into the late-replicating inactive X explains L1 accumulation in this chromosome.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

LINE-1 retrotransposons are frequently active in epithelial tumors. In a new study, Taylor, Wu and colleagues now describe that one of the proteins encoded by LINE-1 elements, ORF1p, is detected in the bloodstream of patients with cancer, and can be used as a noninvasive and multicancer biomarker for diagnosis or treatment monitoring. See related article by Taylor, Wu et al., p. 2532 (7).

Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to precisely insert transposable elements into into the genome of cultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3' end of LINE-1_Cfs (i.e., LINE-1_Cf 3'-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.

Long interspersed element-1 (LINE-1, L1) composes ∼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.

The human LINE-1 retrotransposon has the ability to mobilize into a new genomic location through an intracellular replication cycle. Immunofluorescence and in situ hybridization experiments have been developed to detect subcellular localization of retrotransposition intermediates (i.e., ORF1p, ORF2p, and L1 mRNA). Currently, these protocols are also used to validate the interaction between retrotransposition complex components and potential cellular partners involved in L1 replication. Here, we describe in details methods for the identification of LINE-1 proteins and/or RNA in cells transfected with vectors expressing engineered human LINE-1 elements.

Long INterspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 element amplification protocol (LEAP) assays the ability of L1 ORF2p to reverse transcribe L1 RNA in vitro. Ultracentrifugation or immunoprecipitation is used to isolate L1 ribonucleoprotein particle (RNP) complexes from cultured human cells transfected with an engineered L1 expression construct. The isolated RNPs are incubated with an oligonucleotide that contains a unique sequence at its 5' end and a thymidine-rich sequence at its 3' end. The addition of dNTPs to the reaction allows L1 ORF2p bound to L1 RNA to generate L1 cDNA. The resultant L1 cDNAs then are amplified using polymerase chain reaction (PCR) and the products are visualized by gel electrophoresis. Sequencing the resultant PCR products then allows product verification. The LEAP assay has been instrumental in determining how mutations in L1 ORF1p and ORF2p affect L1 reverse transcriptase (RT) activity. Furthermore, the LEAP assay has revealed that the L1 ORF2p RT can extend a DNA primer with mismatched 3' terminal bases when it is annealed to an L1 RNA template. As the LINE-1 biology field gravitates toward studying cellular proteins that regulate LINE-1, molecular genetic and biochemical approaches such as LEAP, in conjunction with the LINE-1-cultured cell retrotransposition assay, are essential to dissect the molecular mechanism of L1 retrotransposition.

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.

In vitro reverse transcriptase assays have been developed to monitor the presence and activity of ORF2p, an essential protein product of the LINE-1 retrotransposon (L1), in cellular fractions. We describe methods for expression and isolation of L1 ribonucleoprotein particles, and identification of ORF2p reverse transcriptase activity. Two independent methods are described: L1 element amplification protocol (LEAP) and direct L1 extension assay (DLEA). The first method involves cDNA synthesis by primer extension using dNTPs followed by a step of PCR amplification. The second method involves primer extension by incorporation of radiolabeled dTMPs followed by dot-blot or gel separation detection. Finally, we discuss the output and benefits of the two methods.

Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes.

14 LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only 15 the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, 16 reshaping both somatic and germline genomes. Due to their high levels of sequence identity and 17 the existence of many polymorphic insertions absent from the reference genome, the 18 transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here 19 we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used 20 somatic cell lines, and identified transcriptional and epigenetic signatures allowing the 21 unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a 22 very restricted subset of L1HS-Ta loci some being polymorphic among individuals significantly 23 contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct 24 cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, 25 governed by individual-, locus-, and cell-type-specific determinants. 26

Transposable elements comprise more than 45% of the human genome and long interspersed nuclear element 1 (LINE-1 or L1) is the only autonomous mobile element remaining active. Since its identification, it has been proposed that L1 contributes to the mobilization and amplification of other cellular RNAs and more recently, experimental demonstrations of this function has been described for many transcripts such as Alu, a nonautonomous mobile element, cellular mRNAs, or small noncoding RNAs. Detailed examination of the mobilization of various cellular RNAs revealed distinct pathways by which they could be recruited during retrotransposition; template choice or template switching. Here, by analyzing genomic structures and retrotransposition signatures associated with small nuclear RNA (snRNA) sequences, we identified distinct recruiting steps during the L1 retrotransposition cycle for the formation of snRNA-processed pseudogenes. Interestingly, some of the identified recruiting steps take place in the nucleus. Moreover, after comparison to other vertebrate genomes, we established that snRNA amplification by template switching is common to many LINE families from several LINE clades. Finally, we suggest that U6 snRNA copies can serve as markers of L1 retrotransposition dynamics in mammalian genomes.

ABSTRACT Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80–100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.

L1 retrotransposons express proteins (ORF1p and ORF2p) that preferentially mobilize their encoding RNA in cis, but they also can mobilize Alu RNA and, more rarely, cellular mRNAs in trans. Although these RNAs differ in sequence, each ends in a 3' polyadenosine (poly(A)) tract. Here, we replace the L1 polyadenylation signal with sequences derived from a non-polyadenylated long non-coding RNA (MALAT1), which can form a stabilizing triple helix at the 3' end of an RNA. L1/MALAT RNAs accumulate in cells, lack poly(A) tails, and are translated; however, they cannot retrotranspose in cis. Remarkably, the addition of a 16 or 40 base poly(A) tract downstream of the L1/MALAT triple helix restores retrotransposition in cis. The presence of a poly(A) tract also allows ORF2p to bind and mobilize RNAs in trans. Thus, a 3' poly(A) tract is critical for the retrotransposition of sequences that comprise approximately one billion base pairs of human DNA.

Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

LINE-1 (L1) is the most represented sequence of the human genome (17% of the total genomic mass). Moreover, it has been proposed for many years and demonstrated more recently that L1 has contributed to the mobilization of pseudogenes, small non-coding RNAs, such as tRNAs or snRNAs, and SINEs. In fact, it is estimated that L1 is responsible for at least 30% of our genome. The mobilization of non-L1 RNAs can occur in different ways and at different steps of the retrotransposition cycle. Here, by looking at U6 snRNA sequences mobilized by L1, we have observed an ancient repeat sequence derived from U6, present in all primate genomes. We were able to trace its origin in Euarchota genomes, most likely during the divergence of the four orders; Scandentia, Dermoptera, Plesiadapiform (extinct) and Primates.

Mobile elements such as transposons and retrotransposons constitute a substantial fraction of eukaryotic genomes. Such elements represent ~45% of the human genome. Long INterspersed Element-1 (LINE-1 or L1) constitutes about 17% of human DNA and is the only known autonomously active human retrotransposon. L1s are bi-cistronic elements that encode two proteins, ORF1p and ORF2p, which are essential for retrotransposition. These proteins associate with the RNA from which they were translated to form an L1 ribonucleoprotein particle (L1-RNP), which is a hypothesized retrotransposition intermediate. The insertion of a new L1 copy occurs by a mechanism known as target-site primed reverse transcription (TPRT), which is performed by the endonuclease and reverse-transcriptase activities of ORF2p and possibly other cellular factors. About 80 to 100 L1s per human genome retain the ability to retrotranspose by this mechanism, and therefore the ongoing mobility of L1 elements contin! ues to impact the evolution of our genome. Here, we developed new molecular tools to detect retrotransposition-competent L1 RNA and proteins through an epitope-tagging strategy. We used a biochemical approach to identify the 'basal' retrotransposition complex containing L1 RNA, epitope-tagged ORF1p and epitope-tagged ORF2p. We also tested retrotransposition-deficient L1s for their ability to form RNPs and to encode an ORF2p-specific reverse transcriptase activity. Using immunocytochemistry coupled with FISH, we observed a cytoplasmic accumulation of L1 RNPs in perinuclear foci in association with stress granules (SG). Interestingly, we observed that the SG protein markers consistently surrounded the L1 RNP foci. Point mutations in the RNA binding domain of ORF1p decreased the ability of the L1 proteins to form cytoplasmic aggregates, and confirmed the role of this domain in L1 RNP formation. Thus, these results further our understanding of the L1 RNP complex and the general mechanism of L1 retrotransposition.