Paired mutations abolish and restore the balanced annealing and melting activities of ORF1p that are required for LINE-1 retrotransposition

Retrotransposition amplifies LINE-1 (L1) to high copy number in mammalian genomes. The L1 protein encoded by ORF1 (ORF1p) is required for retrotransposition. This dependence on ORF1p was investigated by mutating three highly conserved residues, R238, R284 and Y318 to alanine, thereby inactivating retrotransposition. R284A and Y318A were rescued by further substituting the alanine with the appropriate conservative amino acid, e.g. lysine or phenylalanine, respectively, whereas R238K remained inactive. Quantification of the steady-state levels of L1 RNA and ORF1p failed to discriminate active from inactive variants, indicating loss of L1 retrotransposition resulted from loss of function rather than reduced expression. The two biochemical properties known for ORF1p are high-affinity RNA binding and nucleic acid chaperone activity. Only R238A/K exhibited significantly reduced RNA affinities. The nucleic acid chaperone activities of the remaining paired mutants were assessed by single-molecule DNA stretching and found to mirror retrotransposition activity. To further examine ORF1p chaperone function, their energetic barriers to DNA annealing and melting were derived from kinetic work. When plotted against each other, the ratio of these two activities distinguished functional from non-functional ORF1p variants. These findings enhance our understanding of the requirements for ORF1p in LINE-1 retrotransposition and, more generally, nucleic acid chaperone function.     

Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.     

Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE

Long interspersed elements (LINEs) are mobile elements that comprise a large proportion of many eukaryotic genomes. Although some LINE-encoded open reading frame 1 proteins (ORF1ps) were suggested to be required for LINE mobilization through binding to their RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the esterase-type ORF1p, is especially interesting because it has no known RNA-binding domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated with known ORF1ps, including self-interaction, nucleic acid binding, and nucleic acid chaperone activities. In particular, we showed that its chaperone activity is reversible, suggesting that the chaperone activities of many other ORF1ps are also reversible. From this discovery, we propose that LINE ORF1ps play a general role in LINE integration by forming a complex with LINE RNA and rearranging its conformation.     

Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded beta sheet that is packed against three alpha-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.     

Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.     

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu 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.     

LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.     

Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.     

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.     

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’.     

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes.

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.