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.

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.

Transposable elements (TEs) or "jumping genes" historically have been disparaged as a class of "junk DNA" in mammalian genomes (1,2). The advent of whole genome DNA sequencing, in conjunction with molecular genetic, biochemical, and modern genomic and functional studies, is revealing that TEs are biologically important components of mammalian genomes. TEs are classified by whether they mobilize via a DNA or an RNA intermediate (detailed in (3)). Classical DNA transposons, such as the maize Activator/ Dissociation (Ac/Ds) elements originally discovered by Barbara McClintock, move via a DNA intermediate (4,5). Their mobility (i.e., transposition) can impact organismal phenotypes such as corn kernel variegation. Retrotransposons, the predominant class of TEs in most mammalian genomes, mobilize via an RNA intermediate by a process termed retrotransposition (6).The completion of the human genome reference sequence (HGR) (7, 8) confirmed the results of DNA hybridization-based re-annealing studies (9, 10) and revealed that retrotransposons have been a major force in shaping the structure and function of mammalian genomes. The mobility of non-long terminal repeat (non-LTR) retrotransposons, namely autonomously active Long INterspersed Element-1 sequences (LINE-1s, also known proteins can work efficiently in trans, it is formally possible that trans-complementation might allow the assembly of functional virus-like particles from partially defective HERVs, allowing the generation of new retrotransposition events. Advances in DNA sequencing technologies may reveal rare, active HERV-K elements or de novo germline or somatic HERV-K retrotransposition events in individual human genomes.LTR-retrotransposons are present at greater than 600,000 copies in mouse DNA and comprise approximately 10% of the genome (21). In contrast to the human genome, the mouse genome contains multiple, active ERV subfamilies (reviewed in (50, 51)). These include autonomously active Mus D and intracisternal A particle (IAP) elements, as well as non-autonomous early transposons (ETns) and mammalian apparent LTR retrotransposons (MALRs). It is estimated that ERV insertions are responsible for approximately ten percent of spontaneously arising mouse mutations (reviewed in (51)) (discussed in greater detail in other Chapters of Mobile DNA III). LINE-1 Retrotransposons: abundance and structureA brief overview of human LINE-1 evolution and nomenclature-LINE-1 retrotransposons have been amplifying in mammalian genomes for greater than 160 million years (52-54). In humans, the vast majority of LINE-1 sequences have amplified since the divergence of the ancestral mouse and human lineages approximately 65-75 million years ago (7). As a consequence, LINE-1-derived sequences now account for approximately 17% of human genomic DNA (7) (Figure 1).Sequence comparisons between individual genomic LINE-1 sequences and a consensus sequence derived from modern, active LINE-1s can be used to estimate the age of genomic LINE-1s. These analyses uncovered sixteen LINE-1 pri...