Chapter 51 : The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes

Non-long terminal repeat (LTR) retrotransposons of the human and mouse genomes. The top and bottom panels represent non-LTR retrotransposons in the human and mouse genomes, respectively. Each non-LTR retrotransposon is listed with its name, structure, average size, copy number, percentage of the genome reference sequence occupied by the element, and, if applicable, the active subfamilies (question marks [?] denote uncertainty in whether Alu Sx and SVA-D and F elements are active in vivo). Details of the structure and abbreviations for human and mouse Long INterspersed Element-1 retrotransposons (LINE-1s): Untranslated regions (UTRs) (gray boxes); sense and antisense internal promoters (black arrows); monomeric repeats (white triangles) are followed by an untranslated linker sequence (white box) just upstream of open reading frame 1 (ORF1) in the mouse 5′ UTR; ORF1 (yellow box for human LINE-1; brown box for mouse LINE-1) includes a coiled-coil domain (CC), an RNA recognition motif (RRM), and a C-terminal domain (CTD); inter-ORF spacer (gray box between ORF1 and ORF2); ORF2 (blue boxes) includes endonuclease (EN), reverse transcriptase (RT), and cysteine-rich domains (C); poly (A) tract (An downstream of 3′ UTR). For human Alu: 7SL-derived monomers (orange boxes); RNA polymerase III transcription start site (black arrow) and conserved cis-acting sequences required for transcription (A and B white boxes in left 7SL-derived monomer); adenosine-rich fragment (AAA gray box between left and right 7SL-derived monomers); terminal poly (A) tract (AAAA gray box); variable sized flanking genomic DNA (interrupted small gray box) followed by the RNA pol III termination signal (TTTT). For human SVA: hexameric CCCTCT repeat ((CCCTCT)n light green box); inverted Alu-like repeat (green box with backward arrows); GC-rich VNTR (striped green box); SINE-R sequence sharing homology with HERV-K10, (envelope [ENV] and LTR); cleavage polyadenylation specific factor (CPSF) binding site; terminal poly (A) tract (An). For human and mouse processed pseudogenes: spliced cellular mRNA with UTR (gray boxes) and coding ORF (red boxes for human and purple boxes for mouse, boxes are interrupted by exon–exon junctions [vertical black lines]). For mouse B1 and B2: 7SL-derived monomer (light orange boxes) or tRNA derived sequence (dark orange boxes); RNA pol III transcription start site (black arrow) and conserved cis-acting sequences required for transcription (A and B white boxes); terminal poly (A) tract (AAAA dark gray box); variable sized flanking genomic DNA (interrupted gray box) followed by the RNA polymerase III termination signal (TTTT). The 3′ end of B2 also contains a non-tRNA derived sequence (3′ domain light gray box). Mouse ID and B4 elements are not represented in the figure. References are provided in the text.

Engineered Long INterspersed Element-1 (LINE-1) structure and cell based strategies to study retrotransposition. The LINE-1 expression vector consists of a retrotransposition-competent LINE-1 subcloned into pCEP4 (flanked by a CMV promoter and an SV40 polyadenylation signal). The pCEP4 vector is an episomal plasmid that has protein encoding (EBNA-1) and cis-acting (OriP) sequences necessary for replication in mammalian cells; it also has a hygromycin resistance gene (HYG) that allows for the selection of mammalian cells containing the vector, as well as a bacterial origin of replication (Ori) and ampicillin selection marker (Amp) for plasmid amplification in bacteria. The mneoI reporter cassette, located in the LINE-1 3′ UTR, contains the neomycin phosphotransferase gene (NEO, purple box, with its own promoter and polyadenylation signals, purple arrow and lollipop, respectively) in the opposite transcriptional orientation of LINE-1 transcription. The reporter gene is interrupted by an intron (light purple box) with splice donor (SD) and splice acceptor (SA) sites in the same transcriptional orientation as the LINE-1. This arrangement of the reporter cassette ensures that the reporter gene will only be expressed after a successful round of retrotransposition. De novo retrotransposition of the mneoI reporter cassette will result in G418-resistant colonies that can be quantified—genetic assay panel with pJM101/L1.3 (wild-type [WT]) and pJM105/L1.3 (RT mutant [RT-]) LINE-1 constructs. Alternative reporters can be used instead of mneoI to allow different drug-resistance, fluorescent, or luminescent read-outs (alternative reporters panel, with blasticidin-S deaminase [BLAST], enhanced green fluorescent protein [EGFP] or luciferase [LUC]) retrotransposition indicator cassettes. The addition of the ColE1 bacterial origin of replication (recovery of the insertion panel, green box) to a modified version of the mneoI reporter cassette allows the recovery from cultured cell genomic DNA of engineered LINE-1 retrotransposition events as autonomously replicating plasmids in Escherichia coli. The insertions also can be characterized by inverse polymerase chain reaction using divergent oligonucleotide primers (recovery of the insertion panel, black arrows: 1 and 2) that anneal to the reporter gene. RE indicates restriction enzyme cleavage sites in flanking genomic DNA (gray lines). The use of epitope tags (T7-tag in C-terminus of ORF1, yellow box, and TAP-tag in C-terminus of ORF2, blue box) allow the immunoprecipitation (not shown) and detection of LINE-1 proteins by western blot and immunofluorescence (IF) (detection panel, with western blot data obtained with pAD2TE1, a vector expressing ORF1-T7p and ORF2-TAPp, compared to untransfected [UT] HeLa cells [ 82 ]). The addition of the RNA-stem loops that bind the bacteriophage MS2 coat protein ( 409 ) (orange box) in the 3′ UTR of LINE-1 can be used to detect the cellular localization of LINE-1 RNA by fluorescent in situ hybridization (FISH). Both IF and FISH strategies can be combined to detect the subcellular localization of ORF1p, ORF2p, and LINE-1 RNA (cellular localization panel, with pAD3TE1 vector containing ORF1-T7p, ORF2-TAPp, and LINE-1 RNA-MS2 [ 82 ]). The images shown in the cellular localization and the detection boxes originally were published in ( 82 ). Additional references are provided in the text.

Long INterspersed Element-1 (LINE-1) retrotransposition cycle. An active copy of LINE-1 is present at one chromosomal locus (light blue box in dark gray chromosome) and consists of a 5′ untranslated region (UTR) (light gray box) with an internal promoter (thin black arrow), two open reading frames (ORF1, yellow box, and ORF2, blue box), a 3′ UTR (light gray box) followed by a poly (A) tract (An) and is flanked by target-site duplications (thick black arrows). Transcription of LINE-1 occurs in the nucleus and produces a bicistronic RNA (wavy line). Upon translation in the cytoplasm, ORF1p and ORF2p (yellow circles and blue oval, respectively) bind back to their encoding RNA (cis-preference) to form a ribonucleoprotein particle (RNP) complex. ORF1p and/or ORF2p also can retrotranspose cellular RNAs (mRNA, SVA, and Alu, in red, green, and orange wavy lines, respectively). The retrotransposition of Alu RNA only requires ORF2p ( 92 ). There is some debate as to whether ORF1p augments Alu retrotransposition ( 410 ), and if SVA retrotransposition requires both ORF1p and ORF2p ( 94 , 95 ). The LINE-1 RNP enters the nucleus where de novo insertion occurs by a mechanism termed target-site primed reverse transcription (TPRT). The ORF2p endonuclease activity makes a single-strand endonucleolytic nick at the genomic DNA target (L1 EN cleavage), at a loosely defined consensus site (5′-TTTT/A-3′, with “/” indicating the scissile phosphate). The ORF2p RT activity then uses the exposed 3′-OH group to initiate first-strand LINE-1 cDNA synthesis using the bound RNA as a template. The final steps of TPRT (i.e., top-strand cleavage, second-strand LINE-1 cDNA synthesis, and repair of the DNA ends) lead to the insertion of a de novo LINE-1 copy at a new chromosomal locus (light yellow box in light gray chromosome). The new LINE-1 copy is often 5′ truncated, contains a variable-sized poly (A) tract (An), and generally is flanked by target-site duplications (thick gray arrows). Additional references are provided in the text.

Alterations generated upon Long INterspersed Element-1 (LINE-1) retrotransposition. (A) LINE-1 retrotransposition events can alter target-site genomic DNA. De novo insertion of LINE-1 occurs at a genomic DNA target (thick gray line). LINE-1 RNA (blue wavy line) is followed by a poly (A) tail (An); LINE-1 cDNA (blue arrow); and a new LINE-1 copy (blue box including a poly (A) tract (An)). Insertions can occur by either conventional (full-length, left) or abortive (5′ truncated, right) retrotransposition and generally result in the formation of variable-length target-site duplications (TSD, black boxes). “Twin-priming” generates LINE-1 inversion/deletions or inversion/duplications (represented by opposing arrows in the new LINE-1 copy). The priming of LINE-1 cDNA synthesis from the cleaved top-strand genomic DNA is represented by the light blue arrow. The transduction of genomic DNA sequences can occur when either 5′ or 3′ flanking genomic sequences are incorporated into LINE-1 RNAs and are mobilized by retrotransposition. The 5′ and 3′ transductions are depicted in both LINE-1 RNA (green or pink wavy lines) and the new LINE-1 copy (green or pink boxes). The 3′ transduction events contain two poly (A) sequences (An). The LINE-1 enzymatic machinery also can mobilize small nuclear RNAs (snRNAs) such as U6 snRNA to new genomic locations. The proposed model involves an L1 RT template switch from LINE-1 RNA to the U6 snRNA (orange wavy line) to generate U6 cDNA (orange arrow) during target-site primed reverse transcription (TPRT). (B) LINE-1 retrotransposition events associated with genomic structural variation. LINE-1 RNA, cDNA, and a de novo LINE-1 insertion are depicted as in panel A. Lower case letters (a, b, c, or d) in genomic DNA (gray boxes) are used to depict deletions or duplications (by alteration of the alphabetical order). The resolution of TPRT at the site of DNA damage (left panel, black arrowhead upstream of the integration site) is hypothesized to result in a large genomic deletion (the loss of segment “b”), whereas the resolution of TPRT at a single-strand endonucleolytic nick downstream from the LINE-1 integration site (left panel, black arrowhead) is hypothesized to lead to a large target-site duplication (the duplication of segment “c”). The resolution of TPRT by single-strand annealing (middle) can lead to the generation of a chimeric LINE-1, where an endogenous LINE-1 (light purple box) is fused to a new LINE-1 (dark blue box); the formation of the chimera results in the loss of segment “b”. Similarly, the resolution of “twin-priming” intermediates by synthesis-dependent strand annealing (right) can lead to the generation of an L1 chimera with an intrachromosomal duplication (the duplication of both segment “a” and the endogenous L1 sequence). The entire insertion is flanked by target-site duplications (black boxes). Notably, synthesis-dependent strand annealing can occasionally repair LINE-1 insertions generated in cultured cells by “twin-priming” ( 156 ). Details on how chimeric LINE-1 integration events are formed can be found elsewhere ( 156 , 202 , 203 ). Additional references are provided in the text.

Hypothetical consequences of retrotransposition in pluripotent cells of the early embryo. (A) Cells harboring a de novo retrotransposition event could contribute both to the soma and germline, resulting in an individual with somatic as well as germline mosaicism and a heritable insertion. (B) Conceivably, cells harboring the retrotransposon insertion could contribute solely to the germline, giving rise to germline mosaicism, thereby rendering the insertion heritable. (C) Retrotransposon insertion-bearing cells could contribute to the somatic lineage but not to the germline, resulting in somatic mosaicism. Such an event would not be transmissible to the next generation. Red and white shaded circles in the human figures and sperm represent retrotransposon insertion-bearing and non-insertion-bearing cells in the soma and germline, respectively. (This figure was reproduced from Sandra Richardson’s doctoral thesis [ 408 ]). Additional references are provided in the text.

Long INterspersed Element-1 (LINE-1) retrotransposition in the brain and in cancer. (A) Model for how LINE-1 generates somatic mosaicism in the brain. Sox2, MeCP2, and promoter methylation (red X over the LINE-1 5′ untranslated region [UTR]) are hypothesized to repress LINE-1 expression in neural stem cells (yellow cell). The differentiation of neural stem cells into neuronal precursor cells (NPCs) correlates with a reduction in LINE-1 promoter methylation and a derepression of LINE-1 expression, allowing a permissive milieu for retrotransposition (insertion-bearing NPC [blue cell]). Subsequent differentiation of NPCs into neurons leads to somatic LINE-1 mosaicism in the brain (insertion-bearing neurons [blue cells]). It is unknown whether LINE-1 retrotransposition occurs in postmitotic neurons. (B) Model for how LINE-1 may act as a “driver” or “passenger” mutation during cancer progression. In a somatic cell (yellow cell), LINE-1 expression generally is repressed by promoter methylation (red X over the LINE-1 5′ UTR). After oncogenic transformation (top panel), the derepression of LINE-1 expression in some tumor cells (green cells), allows de novo LINE-1 retrotransposition events that act as “passenger” mutations (insertion-bearing tumor cell in red), leading to somatic mosaicism in the resultant tumor. Alternatively, tumorigenesis can be triggered by a de novo LINE-1 retrotransposition event that acts as a potential “driver” mutation (bottom panel), leading to the clonal amplification of the insertion-bearing cell (red cell). Additional references are provided in the text.