Restricting retrotransposons: a review

Abstract

Retrotransposons have generated about 40 % of the human genome. This review examines the strategies the cell has evolved to coexist with these genomic "parasites", focussing on the non-long terminal repeat retrotransposons of humans and mice. Some of the restriction factors for retrotransposition, including the APOBECs, MOV10, RNASEL, SAMHD1, TREX1, and ZAP, also limit replication of retroviruses, including HIV, and are part of the intrinsic immune system of the cell. Many of these proteins act in the cytoplasm to degrade retroelement RNA or inhibit its translation. Some factors act in the nucleus and involve DNA repair enzymes or epigenetic processes of DNA methylation and histone modification. RISC and piRNA pathway proteins protect the germline. Retrotransposon control is relaxed in some cell types, such as neurons in the brain, stem cells, and in certain types of disease and cancer, with implications for human health and disease. This review also considers potential pitfalls in interpreting retrotransposon-related data, as well as issues to consider for future research.

Keywords: Alu; Autoimmunity; Epigenetics; LINE-1; Methylation; RNAi; Restriction; Retrovirus; SINE; SVA.

Fig. 1

Types of transposable elements in mammals. Abbreviations: DR, direct repeat; ITR, inverted terminal repeat; Gag, group-specific antigen; Prt, protease; Pol, polymerase; Env, envelope; RT, reverse transcriptase domain; INT, integrase domain; TSD, target site duplication; LTR, long terminal repeat; EN, endonuclease domain; C, zinc knuckle domain; A_n, poly (A); A/B, A- and B-box Pol III promoter; SVA, SINE-R, VNTR, Alu element; VNTR, variable number tandem repeats (reproduced from [3]; Elsevier license number 3803340576977)

Fig. 2

Subcellular distribution of LINE-1 ORF1 protein. a. Endogenous ORF1p detected in human embryonal carcinoma 2102Ep cells by a monoclonal antibody [57]. ORF1p is mostly cytoplasmic where it concentrates in SGs and PBs and occasionally at the nuclear membrane. It is faintly detectable in some nuclei and concentrates in nucleoli of a small percentage of cells. Expression of GFP-tagged TDP43 in nuclei but not in nucleoli is shown as a marker. b. Exogenously expressed GFP-tagged ORF1p strongly concentrates at the nuclear membrane and in perinucleolar foci of 5 % or fewer human embryonic kidney (HEK) 293T cells, with attendant reduction in size and number of cytoplasmic granules (left panel). Construct ORF1-EGFP L1-RP contains a CMV promoter, ORF1 C-terminally tagged with EGFP, followed by intact downstream L1 sequence. Nucleoli are marked by α-C23 (nucleolin) antibody (Santa Cruz) and nuclei are stained with Hoechst (right panel)

Fig. 3

Cell culture retrotransposition assay reporter gene cassettes come in a variety of flavors. a. LINE-1 assays. A retrotransposition-competent L1 and reporter cassette is cloned in pCEP4 (Invitrogen)-based vectors, which encode EBNA-1 and OriP and so replicate in primate cells. Variants of the vectors also contain or lack an exogenous promoter upstream of the L1, and encode resistance to hygromycin or puromycin permitting antibiotic selection of transfected cells. mneoI and mblastI reporter cassettes confer drug resistance to cells having a retrotransposition event. These cells are expanded in culture to form colonies, fixed, stained, and the number of colonies scored. The mEGFPI cassette fluorescently marks cells with retrotransposon insertions and allows their numbers to be counted by flow cytomentry. Firefly luciferase gene mFlucI reporter vectors may be cotranfected with pGL4.73 (Promega) or other vector which constitutively expresses renilla luciferase from transfected cells. Following cell lysis, retrotransposition levels, indicated by firefly luciferase, are adjusted to renilla expression to control for differences in transfection efficiency. The mGlucI cassette expresses Gaussia luciferase which when secreted into the media serves as an effective read-out of accumulated retrotransposition events. Levels of Gluc may be normalized to those of Cypridina luciferase (which is also secreted and does not cross-react with Gluc) constiitutively expressed from the cotransfected pSV40-CLuc vector (NEB). Simply by sampling small aliquots of cell culture media, retrotransposition may be assessed in a single well at multiple time points without cell lysis. Luciferase-based reporter cassettes are amenable to HT retrotransposition screening. b. The Alu assay. An active Ya5 Alu and neo^TET cassette interrupted by a Tetrahymena thermophila self-splicing 23S rRNA Group I intron is cloned between the 7SL pol III enhancer and terminator. When this construct is co-expressed with L1 ORF2 alone or a full-length retrotransposition-competent L1, Alu RNAs are reverse transcribed along with the spliced npt gene and integrated into the genome to confer neomycin resistance. Abbreviations: 7SL enh, 7SL enhancer; 7SL TTTT, 7SL transcription terminator; ampR, ampicillin resistance gene; bsd, blasticidin S deaminase gene; CMV, cytomegalovirus promoter; EBNA-1, Epstein-Barr nuclear antigen 1; EGFP, enhanced green fluorescent protein; L mon, left monomer; mini, chimeric mini-intron of the plasmid psiCHECK-2 (Promega); npt, neomycin phosphotransferase gene; oriP, latent origin of replication; pCI: synthetic intron from pCI (Promega); R mon, right monomer; SA, splice acceptor; SD, splice donor; SV40, simian virus 40 early enhancer/promoter; TET, T. thermophila self-splicing intron; TK, herpes simplex virus thymidine kinase poly(A) signal