Retrotransposition Assay Research Articles

Abstract 345: Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype

Abstract Diagnosis of Cutaneous T-Cell Lymphomas (CTCL) can take up to 6 years, increasing the risk of disease progression in these patients. Transcriptomic analyses have helped identify several diagnosis biomarkers, such as TOX, KIRD3DL2, GTSF1 and others. Although not officially a Cancer-Testis (CT) antigen, ectopic expression of Gametocyte Specific Factor 1 (GTSF1), presents the same advantages than CT antigens. Its germline-cell restricted expression potentially makes this gene easily identifiable by the immune system and a therapeutic target with less side effects. The role of GTSF1 has been elucidated in mice, where it controls retrotransposon expression during germ-cell development. Here, we investigate the molecular function of GTSF1 in CTCL. Using shRNA-mediated GTSF1 knockdown in CTCL cell lines, our current work demonstrates an unexpected role of GTSF1 regulating the Th phenotype of CTCL cells. With multiple approaches (qRT-PCR, Western blotting and a dual-luciferase based retrotransposition assay) we concluded that GTSF1 knockdown does not lead to retrotransposon reactivation. Transcriptome analysis with RNA-Seq, followed by differential gene expression analysis with DESeq2 and pathway enrichment analysis with GO, KEGG, Reactome and WikiPathways, suggested an involvement of GTSF1 in the Th phenotype of CTCL cells. Differential gene expression analysis revealed 12,496 differentially expressed genes (p adjusted < 0.05). Pathway enrichment analysis demonstrated an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, GTSF1 knockdown leads to an upregulation of the key cytokines associated with a Th1 phenotype, IFNG and TNF. Disease progression of CTCL is associated with a decrease in Th1 cytokine production and an enhanced Th2 cytokine production by malignant T cells. These findings shed light on the molecular function of GTSF1 as a regulator of the Th phenotype in CTCL and paves the way for the development of a valuable treatment target for CTCL patients. Citation Format: Amelia Martínez Villarreal, Ivan Litvinov. Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 345.

LINE-1 Retrotransposition Assays in Embryonic Stem Cells.

The ongoing mobilization of active non-long terminal repeat (LTR) retrotransposons continues to impact the genomes of most mammals, including humans and rodents. Non-LTR retrotransposons mobilize using an intermediary RNA and a copy-and-paste mechanism termed retrotransposition. Non-LTR retrotransposons are subdivided into long and short interspersed elements (LINEs and SINEs, respectively), depending on their size and autonomy; while active class 1 LINEs (LINE-1s or L1s) encode the enzymatic machinery required to mobilize in cis, active SINEs use the enzymatic machinery of active LINE-1s to mobilize in trans. The mobilization mechanism used by LINE-1s/SINEs was exploited to develop ingenious plasmid-based retrotransposition assays in cultured cells, which typically exploit a reporter gene that can only be activated after a round of retrotransposition. Retrotransposition assays, in cis or in trans, are instrumental tools to study the biology of mammalian LINE-1s and SINEs. In fact, these and other biochemical/genetic assays were used to uncover that endogenous mammalian LINE-1s/SINEs naturally retrotranspose during early embryonic development. However, embryonic stem cells (ESCs) are typically used as a cellular model in these and other studies interrogating LINE-1/SINE expression/regulation during early embryogenesis. Thus, human and mouse ESCs represent an excellent model to understand how active retrotransposons are regulated and how their activity impacts the germline. Here, we describe robust and quantitative protocols to study human/mouse LINE-1 (in cis) and SINE (in trans) retrotransposition using (human and mice) ESCs. These protocols are designed to study the mobilization of active non-LTR retrotransposons in a cellular physiologically relevant context.

462 Gametocyte Specific Factor 1 contributes to the Th phenotype in Cutaneous T-Cell Lymphomas

Ectopic expression of Gametocyte Specific Factor 1 (GTSF1) in Cutaneous T-Cell Lymphoma (CTCL) patients has been reported by several groups. Moreover, it has been associated with a worse prognosis and late-stage CTCL. GTSF1 is a gamete gene that participates in the piRNA pathway to silence retrotransposons on the genome. Despite the established association with a worse prognosis the molecular function of GTSF1 expression remains unknown. Therefore, we established shRNA-mediated GTSF1 knockdown CTCL cell lines. Using qRT-PCR, Western blot and a retrotransposition assay, we demonstrated that GTSF1 knockdown does not lead to mobilization of the retrotransposon LINE-1. These data suggest GTSF1 has a novel role in CTCL. Interestingly, after GTSF1 knockdown in the Mac2A cell line, we observed striking growth inhibition, suggesting this gene is a key player in CTCL carcinogenesis. To further explore this role, we performed RNA-Seq, comparing GTSF1 knockdown to control cells. Differential gene expression analysis using DESeq2, revealed 12496 differentially expressed genes (p adjusted <0.05). Pathway enrichment analysis was performed via GO, KEGG, Reactome and WikiPathways. We observed an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, transcriptome analysis showed an increased expression of cytokine induced genes, such as XAF1, IFI27 and IFI44L, and genes associated with a Th1 phenotype, IFNG and TNF. Research shows that CTCL progression leads to defects in Th1 cytokine production (IFN-γ and TNF-α) by antitumor immune cells and an enhanced Th2 cytokine production (IL-4 and IL-5) by malignant T cells. The chronic production of Th2 cytokines by malignant cells leads to immune deficiency in patients, allowing malignant cells to proliferate and the disease to progress. Taken together, these data suggest GTSF1 as a novel regulator of the Th phenotype on CTCL, thereby placing it as a potential treatment target for regulating disease progression.

Ongoing endeavors to detect mobilization of transposable elements

Transposable elements (TEs) are DNA sequences capable of mobilization from one location to another in the genome. Since the discovery of ‘Dissociation (Dc) locus’ by Barbara McClintock in maize (1), mounting evidence in the era of genomics indicates that a significant fraction of most eukaryotic genomes is composed of TE sequences, involving in various aspects of biological processes such as development, physiology, diseases and evolution. Although technical advances in genomics have discovered numerous functional impacts of TE across species, our understanding of TEs is still ongoing process due to challenges resulted from complexity and abundance of TEs in the genome. In this mini-review, we briefly summarize biology of TEs and their impacts on the host genome, emphasizing importance of understanding TE landscape in the genome. Then, we introduce recent endeavors especially in vivo retrotransposition assays and long read sequencing technology for identifying de novo insertions/TE polymorphism, which will broaden our knowledge of extraordinary relationship between genomic cohabitants and their host.

RNA sensor MDA5 suppresses LINE-1 retrotransposition by regulating the promoter activity of LINE-1 5\u2032-UTR

BackgroundType 1 long interspersed elements, or LINE-1, are the only retroelements that replicate autonomously in human cells. The retrotransposition process of LINE-1 can trigger the activation of the innate immune system and has been proposed to play a role in the development of several autoimmune diseases, including Aicardi-Goutières syndrome (AGS). In contrast, all known AGS-associated proteins, except MDA5, have been reported to affect LINE-1 activity. Thus, MDA5 is likely to also function as a LINE-1 suppressor.ResultsMDA5 was found to potently suppress LINE-1 activity in a reporter-based LINE-1 retrotransposition assay. Although MDA5 is an endogenous RNA sensor able to activate the innate immune system, increased interferon (IFN) expression only contributed in part to MDA5-mediated LINE-1 suppression. Instead, MDA5 potently regulated the promoter activity of LINE-1 5′-UTR, as confirmed by transiently expressed myc-tagged MDA5 or knockdown of endogenous MDA5 expression. Consequently, MDA5 effectively reduced the generation of LINE-1 RNA and the subsequent expression of LINE-1 ORF1p and ORF2p. Interestingly, despite MDA5 being a multi-domain protein, the N-terminal 2CARD domain alone is sufficient to interact with LINE-1 5′-UTR and inhibit LINE-1 promoter activity.ConclusionOur data reveal that MDA5 functions as a promoter regulator; it directly binds to the LINE-1 5′-UTR and suppresses its promoter activity. Consequently, MDA5 reduces LINE-1 RNA and protein levels, and ultimately inhibits LINE-1 retrotransposition. In contrast, MDA5-induced IFN expression only plays a mild role in MDA5-mediated LINE-1 suppression. In addition, the N-terminal 2CARD domain was found to be a functional region for MDA5 upon inhibition of LINE-1 replication. Thus, our data suggest that besides being an initiator of the innate immune system, MDA5 is also an effector against LINE-1 activity, potentially forming a feedback loop by suppressing LINE-1-induced innate immune activation.

HCV Activates Somatic L1 Retrotransposition-A Potential Hepatocarcinogenesis Pathway.

Simple SummaryChronic hepatitis C virus (HCV) infection is a common cause of liver cancer in the developed world. Although anti-viral treatment can cure HCV infection, the risk of cancer development remains in individuals with consequent advanced fibrosis or cirrhosis. In this study, we show that HCV can influence transposable DNA elements called L1 retrotransposons. These are mobile genetic elements that can negatively alter the host genome, potentially promoting cancer development. L1 elements are known to be activated in several cancer types, including liver cancer. Hence, we suggest a novel pathway involved in liver cancer development in patients with chronic HCV, including those with active infection as well as after viral clearance.Hepatitis C virus (HCV) is a common cause of hepatocellular carcinoma (HCC). The activation and mutagenic consequences of L1 retrotransposons in virus-associated-HCC have been documented. However, the direct influence of HCV upon L1 elements is unclear, and is the focus of the present study. L1 transcript expression was evaluated in a publicly available liver tissue RNA-seq dataset from patients with chronic HCV hepatitis (CHC), as well as healthy controls. L1 transcript expression was significantly higher in CHC than in controls. L1orf1p (a L1 encoded protein) expression was observed in six out of 11 CHC livers by immunohistochemistry. To evaluate the influence of HCV on retrotransposition efficiency, in vitro engineered-L1 retrotransposition assays were employed in Huh7 cells in the presence and absence of an HCV replicon. An increased retrotransposition rate was observed in the presence of replicating HCV RNA, and persisted in cells after viral clearance due to sofosbuvir (PSI7977) treatment. Increased retrotransposition could be due to dysregulation of the DNA-damage repair response, including homologous recombination, due to HCV infection. Altogether these data suggest that L1 expression can be activated before oncogenic transformation in CHC patients, with HCV-upregulated retrotransposition potentially contributing to HCC genomic instability and a risk of transformation that persists post-viral clearance.

The RNA editing enzyme ADAR2 restricts L1 mobility

ABSTRACT Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex. Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

Characterization of an active LINE-1 in the naked mole-rat genome

Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-living rodent species. A reason for their long lifespan is pronounced cancer resistance. Therefore, researchers believe that NMRs have unknown secrets of cancer resistance and seek to find them. Here, to reveal the secrets, we noticed a retrotransposon, long interspersed nuclear element 1 (L1). L1s can amplify themselves and are considered endogenous oncogenic mutagens. Since the NMR genome contains fewer L1-derived sequences than other mammalian genomes, we reasoned that the retrotransposition activity of L1s in the NMR genome is lower than those in other mammalian genomes. In this study, we successfully cloned an intact L1 from the NMR genome and named it NMR-L1. An L1 retrotransposition assay using the NMR-L1 reporter revealed that NMR-L1 was active retrotransposon, but its activity was lower than that of human and mouse L1s. Despite lower retrotrasposition activity, NMR-L1 was still capable of inducing cell senescence, a tumor-protective system. NMR-L1 required the 3′ untranslated region (UTR) for retrotransposition, suggesting that NMR-L1 is a stringent-type of L1. We also confirmed the 5′ UTR promoter activity of NMR-L1. Finally, we identified the G-quadruplex structure of the 3′ UTR, which modulated the retrotransposition activity of NMR-L1. Taken together, the data indicate that NMR-L1 retrotranspose less efficiently, which may contribute to the cancer resistance of NMRs.

SRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay.

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

Long interspersed element-1 (LINE-1 or L1) is the only autonomous retrotransposon active in human cells. Different host factors have been shown to influence L1 mobility however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the Double-Stranded Break (DSB) repair and Fanconi Anemia factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and also plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a “battleground” at the DNA replication fork between HR factors and L1 retrotransposons, and revealing a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.

A study of meiomitosis and novel pathways of genomic instability in cutaneous T-cell lymphomas (CTCL).

Genomic instability is a hallmark of cancer and an enabling factor for genetic alterations that drive cancer development and progression. The clashing of mitosis and aberrantly expressed meiosis machineries, which may contribute to genomic instability, has been coined cancer “meiomitosis”. LINE-1 retrotransposition, a process active in germ cells, acts outside of the meiotic machinery to create DNA double strand breaks (DNA DSBs) and has played an important role in the evolution of the human genome. We have previously demonstrated that in CTCL several cancer testis/meiotic genes are expressed. Furthermore, this cancer exhibits extensive and ongoing chromosomal/microsatellite instability. In this study we analyzed immortalized patient-derived cells and primary CTCL patient samples using RT-PCR, western blotting and confocal microscopy and found that proteins critically involved in meiosis and LINE-1 retrotransposition are expressed and are associated with chromosomal instability and DNA DSB formation. Using cell cycle synchronization, we show G1/S phase-transition-specific expression of meiosis proteins. Using the Alu retrotransposition assay, we demonstrate the functional activity of LINE-1 retrotransposon in CTCL. Histone acetyltransferase inhibition results in downregulation of the ectopic germ cell programs and concomitant decrease in DNA DSBs foci formation. Notably, LINE-1 and meiosis genes were expressed across a panel of other solid tumor cell lines. Taken together, our results indicate that malignant cells in culture undergo “cancer meiomitosis” rather than the classic mitosis division. The ectopic expression of meiosis genes and reactivation of LINE-1 may be contributing to genomic instability and represent novel targets for immunotherapy in this and other cancers.

LTR-Retrotransposon Control by tRNA-Derived Small RNAs

Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control.

Heritable L1 retrotransposition in the mouse primordial germline and early embryo

LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo.

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition.

Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80–100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.

ADAR1 restricts LINE-1 retrotransposition.

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR).

Long Interspersed Element 1 (LINE-1 or L1) is capable of causing genomic instability through the activity of the L1 ORF2 protein (ORF2p). This protein contains endonuclease (EN) and reverse transcriptase (RT) domains that are necessary for the retrotransposition of L1 and the Short Interspersed Element (SINE) Alu. The functional importance of approximately 50% of the ORF2p molecule remains unknown, but some of these sequences could play a role in retrotransposition, or be necessary for the enzymatic activities of the EN and/or RT domains. Conventional approaches using the full-length, contiguous ORF2p make it difficult to study the involvement of these unannotated sequences in the function of L1 ORF2p. Our lab has developed a Bipartile Alu Retrotransposition (BAR) assay that relies on separate truncated ORF2p fragments: an EN-containing and an RT-containing fragment. We validated the utility of this method for studying the ORF2p function in retrotransposition by assessing the effect of expression levels and previously characterized mutations on BAR. Using BAR, we identified two pairs of amino acids important for retrotransposition, an FF and a WD. The WD appears to play a role in cDNA synthesis by the ORF2p molecule, despite being outside the canonical RT domain.

LINE-1 Cultured Cell Retrotransposition Assay.

The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

LEAP: L1 Element Amplification Protocol.

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.

SINE Retrotransposition: Evaluation of Alu Activity and Recovery of De Novo Inserts.

Mobile element activity is of great interest due to its impact on genomes. However, the types of mobile elements that inhabit any given genome are remarkably varied. Among the different varieties of mobile elements, the Short Interspersed Elements (SINEs) populate many genomes, including many mammalian species. Although SINEs are parasites of Long Interspersed Elements (LINEs), SINEs have been highly successful in both the primate and rodent genomes. When comparing copy numbers in mammals, SINEs have been vastly more successful than other nonautonomous elements, such as the retropseudogenes and SVA. Interestingly, in the human genome the copy number of Alu (a primate SINE) outnumbers LINE-1 (L1) copies 2 to 1. Estimates suggest that the retrotransposition rate for Alu is tenfold higher than LINE-1 with about 1 insert in every twenty births. Furthermore, Alu-induced mutagenesis is responsible for the majority of the documented instances of human retroelement insertion-induced disease. However, little is known on what contributes to these observed differences between SINEs and LINEs. The development of an assay to monitor SINE retrotransposition in culture has become an important tool for the elucidation of some of these differences. In this chapter, we present details of the SINE retrotransposition assay and the recovery of de novo inserts. We also focus on the nuances that are unique to the SINE assay.