ZCCHC3 is a stress granule zinc knuckle protein that strongly suppresses LINE-1 retrotransposition.

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control.

Retrotransposons revisited: the restraint and rehabilitation of parasites.

Retrotransposons, mainly LINEs, SINEs, and endogenous retroviruses, make up roughly 40% of the mammalian genome and have played an important role in genome evolution. Their prevalence in genomes reflects a delicate balance between their further expansion and the restraint imposed by the host. In any human genome only a small number of LINE1s (L1s) are active, moving their own and SINE sequences into new genomic locations and occasionally causing disease. Recent insights and new technologies promise answers to fundamental questions about the biology of transposable elements.

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.

A potential new mechanism for pregnancy loss: considering the role of LINE-1 retrotransposons in early spontaneous miscarriage.

LINE1 retrotransposons are mobile DNA elements that copy and paste themselves into new sites in the genome. To ensure their evolutionary success, heritable new LINE-1 insertions accumulate in cells that can transmit genetic information to the next generation (i.e., germ cells and embryonic stem cells). It is our hypothesis that LINE1 retrotransposons, insertional mutagens that affect expression of genes, may be causal agents of early miscarriage in humans. The cell has evolved various defenses restricting retrotransposition-caused mutation, but these are occasionally relaxed in certain somatic cell types, including those of the early embryo. We predict that reduced suppression of L1s in germ cells or early-stage embryos may lead to excessive genome mutation by retrotransposon insertion, or to the induction of an inflammatory response or apoptosis due to increased expression of L1-derived nucleic acids and proteins, and so disrupt gene function important for embryogenesis. If correct, a novel threat to normal human development is revealed, and reverse transcriptase therapy could be one future strategy for controlling this cause of embryonic damage in patients with recurrent miscarriages.

Aicardi-Goutières syndrome protein TREX1 suppresses L1 and maintains genome integrity through exonuclease-independent ORF1p depletion.

Maintaining genome integrity is important for cells and damaged DNA triggers autoimmunity. Previous studies have reported that Three-prime repair exonuclease 1(TREX1), an endogenous DNA exonuclease, prevents immune activation by depleting damaged DNA, thus preventing the development of certain autoimmune diseases. Consistently, mutations in TREX1 are linked with autoimmune diseases such as systemic lupus erythematosus, Aicardi-Goutières syndrome (AGS) and familial chilblain lupus. However, TREX1 mutants competent for DNA exonuclease activity are also linked to AGS. Here, we report a nuclease-independent involvement of TREX1 in preventing the L1 retrotransposon-induced DNA damage response. TREX1 interacted with ORF1p and altered its intracellular localization. Furthermore, TREX1 triggered ORF1p depletion and reduced the L1-mediated nicking of genomic DNA. TREX1 mutants related to AGS were deficient in inducing ORF1p depletion and could not prevent L1-mediated DNA damage. Therefore, our findings not only reveal a new mechanism for TREX1-mediated L1 suppression and uncover a new function for TREX1 in protein destabilization, but they also suggest a novel mechanism for TREX1-mediated suppression of innate immune activation through maintaining genome integrity.

The Broad-Spectrum Antiviral Protein ZAP Restricts Human Retrotransposition.

Intrinsic immunity describes the set of recently discovered but poorly understood cellular mechanisms that specifically target viral pathogens. Their discovery derives in large part from intensive studies of HIV and SIV that revealed restriction factors acting at various stages of the retroviral life cycle. Recent studies indicate that some factors restrict both retroviruses and retrotransposons but surprisingly in ways that may differ. We screened known interferon-stimulated antiviral proteins previously untested for their effects on cell culture retrotransposition. Several factors, including BST2, ISG20, MAVS, MX2, and ZAP, showed strong L1 inhibition. We focused on ZAP (PARP13/ZC3HAV1), a zinc-finger protein that targets viruses of several families, including Retroviridae, Tiloviridae, and Togaviridae, and show that ZAP expression also strongly restricts retrotransposition in cell culture through loss of L1 RNA and ribonucleoprotein particle integrity. Association of ZAP with the L1 ribonucleoprotein particle is supported by co-immunoprecipitation and co-localization with ORF1p in cytoplasmic stress granules. We also used mass spectrometry to determine the protein components of the ZAP interactome, and identified many proteins that directly interact and colocalize with ZAP, including MOV10, an RNA helicase previously shown to suppress retrotransposons. The detection of a chaperonin complex, RNA degradation proteins, helicases, post-translational modifiers, and components of chromatin modifying complexes suggest mechanisms of ZAP anti-retroelement activity that function in the cytoplasm and perhaps also in the nucleus. The association of the ZAP ribonucleoprotein particle with many interferon-stimulated gene products indicates it may be a key player in the interferon response.

Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition.

LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic 'parasites'.

MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.

Retrotransposition of marked SVA elements by human L1s in cultured cells.

Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed.

A mouse model of human L1 retrotransposition.

The L1 retrotransposon has had an immense impact on the size and structure of the human genome through a variety of mechanisms, including insertional mutagenesis. To study retrotransposition in a living organism, we created a mouse model of human L1 retrotransposition. Here we show that L1 elements can retrotranspose in male germ cells, and that expression of a human L1 element under the control of its endogenous promoter is restricted to testis and ovary. In the mouse line with the highest level of L1 expression, we found two de novo L1 insertions in 135 offspring. Both insertions were structurally indistinguishable from natural endogenous insertions. This suggests that an individual L1 element can have substantial mutagenic potential. In addition to providing a valuable in vivo model of retrotransposition in mammals, these mice are an important step in the development of a new random mutagenesis system.

Discrete subcellular partitioning of human retrotransposon RNAs despite a common mechanism of genome insertion.

Despite the immense significance retrotransposons have had for genome evolution much about their biology is unknown, including the processes of forming their ribonucleoprotein (RNP) particles and transporting them about the cell. Suppression of retrotransposon expression, together with the presence of retrotransposon sequence within numerous mRNAs, makes tracking endogenous L1 RNP particles in cells problematic. We overcame these difficulties by assaying in living and fixed cells tagged-RNPs generated from constructs expressing retrotransposition-competent L1s. In this way, we demonstrate for the first time the subcellular colocalization of L1 RNA and proteins ORF1p and ORF2p, and show their targeting together to cytoplasmic foci. Foci are often associated with markers of cytoplasmic stress granules. Furthermore, mutation analyses reveal that ORF1p can direct L1 RNP distribution within the cell. We also assayed RNA localization of the non-autonomous retrotransposons Alu and SVA. Despite a requirement for the L1 integration machinery, each manifests unique features of subcellular RNA distribution. In nuclei Alu RNA forms small round foci partially associated with marker proteins for coiled bodies, suborganelles involved in the processing of non-coding RNAs. SVA RNA patterning is distinctive, being cytoplasmic but without prominent foci and concentrated in large nuclear aggregates that often ring nucleoli. Such variability predicts significant differences in the life cycles of these elements.

LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex.

LINE-1 retrotransposons constitute one-fifth of human DNA and have helped shape our genome. A full-length L1 encodes a 40-kDa RNA-binding protein (ORF1p) and a 150-kDa protein (ORF2p) with endonuclease and reverse transcriptase activities. ORF1p is distinctive in forming large cytoplasmic foci, which we identified as cytoplasmic stress granules. A phylogenetically conserved central region of the protein is critical for wild-type localization and retrotransposition. Yeast two-hybrid screens revealed several RNA-binding proteins that coimmunoprecipitate with ORF1p and colocalize with ORF1p in foci. Two of these proteins, YB-1 and hnRNPA1, were previously reported in stress granules. We identified additional proteins associated with stress granules, including DNA-binding protein A, 9G8, and plasminogen activator inhibitor RNA-binding protein 1 (PAI-RBP1). PAI-RBP1 is a homolog of VIG, a part of the Drosophila melanogaster RNA-induced silencing complex (RISC). Other RISC components, including Ago2 and FMRP, also colocalize with PAI-RBP1 and ORF1p. We suggest that targeting ORF1p, and possibly the L1 RNP, to stress granules is a mechanism for controlling retrotransposition and its associated genetic and cellular damage.

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease.

A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS.

Improved ThwaRTing of Genome Symbionts.

In this issue of Cell Chemical Biology, Banuelos-Sanchez et al. (2019) present a comprehensive analysis of selective non-toxic inhibitors of reverse transcriptases encoded by endogenous retrotransposons. This work offers tools for the study of these retroelements, whose activity has been linked to cancer, neurological disorders, autoimmunity, and genomic instability.

A human endogenous retrovirus encoded protease potentially cleaves numerous cellular proteins.

BACKGROUND: A considerable portion of the human genome derives from retroviruses inherited over millions of years. Human endogenous retroviruses (HERVs) are usually severely mutated, yet some coding-competent HERVs exist. The HERV-K(HML-2) group includes evolutionarily young proviruses that encode typical retroviral proteins. HERV-K(HML-2) has been implicated in various human diseases because transcription is often upregulated and some of its encoded proteins are known to affect cell biology. HERV-K(HML-2) Protease (Pro) has received little attention so far, although it is expressed in some disease contexts and other retroviral proteases are known to process cellular proteins. RESULTS: We set out to identify human cellular proteins that are substrates of HERV-K(HML-2) Pro employing a modified Terminal Amine Isotopic Labeling of Substrates (TAILS) procedure. Thousands of human proteins were identified by this assay as significantly processed by HERV-K(HML-2) Pro at both acidic and neutral pH. We confirmed cleavage of a majority of selected human proteins in vitro and in co-expression experiments in vivo. Sizes of processing products observed for some of the tested proteins coincided with product sizes predicted by TAILS. Processed proteins locate to various cellular compartments and participate in diverse, often disease-relevant cellular processes. A limited number of HERV-K(HML-2) reference and non-reference loci appears capable of encoding active Pro. CONCLUSIONS: Our findings from an approach combining TAILS with experimental verification of candidate proteins in vitro and in cultured cells suggest that hundreds of cellular proteins are potential substrates of HERV-K(HML-2) Pro. It is therefore conceivable that even low-level expression of HERV-K(HML-2) Pro affects levels of a diverse array of proteins and thus has a functional impact on cell biology and possible relevance for human diseases. Further studies are indicated to elucidate effects of HERV-K(HML-2) Pro expression regarding human substrate proteins, cell biology, and disease. The latter also calls for studies on expression of specific HERV-K(HML-2) loci capable of encoding active Pro. Endogenous retrovirus-encoded Pro activity may also be relevant for disease development in species other than human.

Mobile DNA in Endocrinology: LINE-1 Retrotransposon Causing Partial Androgen Insensitivity Syndrome.

CONTEXT: Androgen insensitivity syndrome (AIS) is the most common cause of disorders of sex development in 46,XY individuals. It is an X-linked condition usually caused by pathogenic allelic variants in the androgen receptor (AR) gene. The phenotype depends on the AR variant, ranging from severe undervirilization (complete AIS) to several degrees of external genitalia undervirilization. Although 90% of those with complete AIS will have AR mutations, this will only be true for 40% of those with partial AIS (PAIS). OBJECTIVE: To identify the genetic etiology of AIS in a large multigenerational family with the PAIS phenotype. PARTICIPANTS: Nine affected individuals with clinical and laboratory findings consistent with PAIS and a normal exonic AR sequencing. SETTINGS: Endocrine clinic and genetic institute from two academic referral centers. DESIGN: Analysis of whole exons of the AR gene, including splicing regions, was performed, followed by sequencing of the 5'untranslated region (UTR) of the AR gene. Detailed phenotyping was performed at the initial diagnosis and long-term follow-up, and circulating levels of steroid gonadal hormones were measured in all affected individuals. AR expression was measured using RT-PCR and cultured fibroblasts. RESULTS: All 46,XY family members with PAIS had inherited, in hemizygosity, a complex defect (∼1100 bp) in the 5'UTR region of the AR surrounded by a duplicated 18-bp sequence (target site duplication). This sequence is 99.7% similar to an active, long, interspersed element present on the X chromosome (AC002980; Xq22.2), which was inserted in the 5'UTR of the AR gene, severely reducing AR expression and leading to PAIS. CONCLUSION: The molecular diagnosis of PAIS remains challenging. The genomic effect of retrotransposon mobilization should be considered a possible molecular cause of AIS and other AR diseases.

Meeting report: mobile genetic elements and genome plasticity 2018.

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11-15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements.

Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. About 90% of ALS cases are without a known genetic cause. The human endogenous retrovirus multi-copy HERV-K(HML-2) group was recently reported to potentially contribute to neurodegeneration and disease pathogenesis in ALS because of transcriptional upregulation and toxic effects of HML-2 Envelope (Env) protein. Env and other proteins are encoded by some transcriptionally active HML-2 loci. However, more detailed information is required regarding which HML-2 loci are transcribed in ALS, which of their proteins are expressed, and differences between the disease and non-disease states. METHODS: For brain and spinal cord tissue samples from ALS patients and controls, we identified transcribed HML-2 loci by generating and mapping HML-2-specific cDNA sequences. We predicted expression of HML-2 env gene-derived proteins based on the observed cDNA sequences. Furthermore, we determined overall HML-2 transcript levels by RT-qPCR and investigated presence of HML-2 Env protein in ALS and control tissue samples by Western blotting. RESULTS: We identified 24 different transcribed HML-2 loci. Some of those loci are transcribed at relatively high levels. However, significant differences in HML-2 loci transcriptional activities were not seen when comparing ALS and controls. Likewise, overall HML-2 transcript levels, as determined by RT-qPCR, were not significantly different between ALS and controls. Indeed, we were unable to detect full-length HML-2 Env protein in ALS and control tissue samples despite reasonable sensitivity. Rather our analyses suggest that a number of HML-2 protein variants other than full-length Env may potentially be expressed in ALS patients. CONCLUSIONS: Our results expand and refine recent publications on HERV-K(HML-2) and ALS. Some of our results are in conflict with recent findings and call for further specific analyses. Our profiling of HML-2 transcription in ALS opens up the possibility that HML-2 proteins other than canonical full-length Env may have to be considered when studying the role of HML-2 in ALS disease.

Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving loss of motor neurons and having no known cure and uncertain etiology. Several studies have drawn connections between altered retrotransposon expression and ALS. Certain features of the LINE-1 (L1) retrotransposon-encoded ORF1 protein (ORF1p) are analogous to those of neurodegeneration-associated RNA-binding proteins, including formation of cytoplasmic aggregates. In this study we explore these features and consider possible links between L1 expression and ALS. RESULTS: We first considered factors that modulate aggregation and subcellular distribution of LINE-1 ORF1p, including nuclear localization. Changes to some ORF1p amino acid residues alter both retrotransposition efficiency and protein aggregation dynamics, and we found that one such polymorphism is present in endogenous L1s abundant in the human genome. We failed, however, to identify CRM1-mediated nuclear export signals in ORF1p nor strict involvement of cell cycle in endogenous ORF1p nuclear localization in human 2102Ep germline teratocarcinoma cells. Some proteins linked with ALS bind and colocalize with L1 ORF1p ribonucleoprotein particles in cytoplasmic RNA granules. Increased expression of several ALS-associated proteins, including TAR DNA Binding Protein (TDP-43), strongly limits cell culture retrotransposition, while some disease-related mutations modify these effects. Using quantitative reverse transcription PCR (RT-qPCR) of ALS tissues and reanalysis of publicly available RNA-Seq datasets, we asked if changes in expression of retrotransposons are associated with ALS. We found minimal altered expression in sporadic ALS tissues but confirmed a previous report of differential expression of many repeat subfamilies in C9orf72 gene-mutated ALS patients. CONCLUSIONS: Here we extended understanding of the subcellular localization dynamics of the aggregation-prone LINE-1 ORF1p RNA-binding protein. However, we failed to find compelling evidence for misregulation of LINE-1 retrotransposons in sporadic ALS nor a clear effect of ALS-associated TDP-43 protein on L1 expression. In sum, our study reveals that the interplay of active retrotransposons and the molecular features of ALS are more complex than anticipated. Thus, the potential consequences of altered retrotransposon activity for ALS and other neurodegenerative disorders are worthy of continued investigation.

Restricting retrotransposons: a review.

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.