A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity

L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, T(FC) and T(Fspa), to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in T(FC), indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity.     

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.     

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.     

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.     

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.     

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.     

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.     

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.     

The RAGNYA fold: a novel fold with multiple topological variants found in functionally diverse nucleic acid, nucleotide and peptide-binding proteins

Using sensitive structure similarity searches, we identify a shared alpha+beta fold, RAGNYA, principally involved in nucleic acid, nucleotide or peptide interactions in a diverse group of proteins. These include the Ribosomal proteins L3 and L1, ATP-grasp modules, the GYF domain, DNA-recombination proteins of the NinB family from caudate bacteriophages, the C-terminal DNA-interacting domain of the Y-family DNA polymerases, the uncharacterized enzyme AMMECR1, the siRNA silencing repressor of tombusviruses, tRNA Wybutosine biosynthesis enzyme Tyw3p, DNA/RNA ligases and related nucleotidyltransferases and the Enhancer of rudimentary proteins. This fold exhibits three distinct circularly permuted versions and is composed of an internal repeat of a unit with two-strands and a helix. We show that despite considerable structural diversity in the fold, its representatives show a common mode of nucleic acid or nucleotide interaction via the exposed face of the sheet. Using this information and sensitive profile-based sequence searches: (1) we predict the active site, and mode of substrate interaction of the Wybutosine biosynthesis enzyme, Tyw3p, and a potential catalytic role for AMMECR1. (2) We provide insights regarding the mode of nucleic acid interaction of the NinB proteins, and the evolution of the active site of classical ATP-grasp enzymes and DNA/RNA ligases. (3) We also present evidence for a bacterial origin of the GYF domain and propose how this version of the fold might have been utilized in peptide interactions in the context of nucleoprotein complexes.     

First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.     

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (≥2 μM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na⁺ and Mg²⁺ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions. © 1997 John Wiley & Sons, Inc.