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PubMed1993

Replacement of His23 by Cys in a zinc finger of HIV-1 NCp7 led to a change in 1H NMR-derived 3D structure and to a loss of biological activity.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which is necessary for the formation of infectious virions, contains two zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form. To elucidate the importance of this particular motif, well conserved in retroviruses and retroelements, we substituted the histidine residue by a cysteine in the first zinc binding domain 13VKCFNCGKEGHTARNCRA30. The structures of the mutated and native zinc complexed peptides were studied by two-dimensional 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution. The nuclear Overhauser effects were used as constraints to determine the solution structures using DIANA software followed by AMBER energy refinement. The results show that native and mutant peptides fold into non-identical three-dimensional structures, probably accounting for the loss of retrovirus infectivity following the His-Cys point mutation.

Amino Acid Sequence↗

PubMed1994

Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon. New evidence for retroviral nature of the gypsy element.

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.

Animals↗

PubMed1990

A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria.

We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.

Base Sequence↗

PubMed1993

Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria.

Group II introns al1 and al2 of the yeast mtDNA cox1 gene encode reverse transcriptase-like proteins that function in RNA splicing and may play a role in intron mobility and excision. We find that ribonucleoprotein particles from yeast mitochondria contain a reverse transcriptase activity that is likely encoded by al1 and al2 and is highly specific for the introns and their flanking exons. Using a mutant strain with elevated activity, we show that the reverse transcriptase uses either excised intron RNA or cox1 pre-mRNA as template and initiates cDNA synthesis near the 3' end of al2 and immediately downstream in E3. Our results suggest that introns al1 and al2 are retroelements, which encode reverse transcriptases that have adapted to function in RNA splicing.

Base Sequence↗

PubMed1992

Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons.

The element; Ty4 is a retrotransposon present in low copy number in the genome of Saccharomyces cerevisiae [Stucka et al., Nucleic Acids Res. 17 (1989) 4993-5001]. We have determined the complete nucleotide sequence of one such element from a particular strain and compared it to the other two elements occurring in this strain. The genomic organization of Ty4 is homologous to that found in other retrotransposons of the Ty1-copia group. The internal part of the element contains two large open reading frames (TY4A and TY4B) overlapping by 226 bp in a + 1 mode. TY4A reveals characteristics of the gag portion of retrotransposons and retroviruses, while TY4B consists of a protease, an integrase, a reverse transcriptase, and an RNase H domain (in that order). Our analyses suggest that only one of these copies might be transpositionally active. Sequence comparisons at the amino acid level show that the domains in Ty4 diverge considerably from those of other retrotransposons. The greatest similarity is seen between the reverse transcriptases (50%), the proteases (40%), and the integrases (30%) of Ty4, Ty1/2 and copia, respectively, whereas the degree of similarity for all other entities of these elements is much lower. Considering evolutionary aspects of the retrotransposons, we have to conclude that Ty4 has diverged at an early stage from the progenitors of other known retroelements and represents a novel and independent subgroup of the Ty1-copia class of retrotransposons.

Amino Acid Sequence↗

PubMed2025

Biochemical assays for AID/APOBECs and the identification of AID/APOBEC inhibitors.

Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.

Cytidine Deaminase↗

PubMed2004

'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA synthesis in human immunodeficiency virus.

During the course of reverse transcription, human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) initiates plus-strand DNA synthesis from two highly conserved, purine-rich RNA segments of the viral genome referred to as the 3' and central polypurine tracts (3' and cPPTs). Processing of these elements occurs in several sequential steps including (1) minus-strand DNA synthesis over the PPT(s), (2) ribonuclease H (RNase H) mediated cleavage at the PPT 3' terminus, (3) plus-strand DNA synthesis from the nascent RNA primer(s), and (4) primer removal. Completing each of these steps precisely and specifically is essential, as failure to do so can result in reduced virus replication and/or impaired integration of viral DNA into the host cell genome. In this review, plus-strand primer processing in HIV-1 is discussed from biochemical, structural, and historical perspectives. A comparative analysis of PPT-processing in different LTR-containing retroelements is also presented.

DNA, Viral↗

PubMed2005

Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation.

All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.

Arabidopsis↗

PubMed2005

RNAi-mediated allelic trans-interaction at the imprinted Rtl1/Peg11 locus.

The Dlk1-Gtl2 imprinted domain, encompassing the callipyge (CLPG) locus in sheep, has recently been shown to harbor a large number of maternally expressed miRNA genes [1, 2]. Two of these (mir127 and mir136) are processed from a transcript (antiPeg11) that is antisense to Rtl1/Peg11, a paternally expressed intronless gene with homology to the gag and pol polyproteins of Sushi-like retroelements [3]. We herein demonstrate that several additional miRNAs are processed from antiPeg11 and that these regulate Rtl1/Peg11 in trans by guiding RISC-mediated cleavage of its mRNA. This is the first demonstration of miRNA-mediated RNAi involving imprinted genes in mammals.

Animals↗

PubMed2004

A processed pseudogene contributes to apparent mule deer prion gene heterogeneity.

Pathogenesis and transmission of the prion disorders (transmissible spongiform encephalopathies, TSEs) are mediated by a modified isoform of the prion protein (PrP). Prion protein gene (PRNP) alleles associated with relative susceptibility to TSE have been identified in sheep, humans and possibly elk. Comparable data have not been derived for mule deer, a species susceptible to the TSE chronic wasting disease (CWD). Initial analysis of the open reading frame (ORF) in exon 3 of the mule deer PRNP gene revealed polymorphisms in all 145 samples analyzed, with 10 potential polymorphic sites. Because 144/145 (99.3%) of the samples were heterozygous for a coding change (N/S) at codon 138 (bp 412) and a non-coding polymorphism at bp 418, and individual deer with three or four different alleles were identified a possible gene duplication was indicated. Analysis of BAC clones containing mule deer PRNP genes revealed a full length functional gene and a processed pseudogene. The pseudogene was characteristic of previously described retroelements, in that it lacks introns and is flanked by repeat sequences. Three alleles of the functional gene were identified, with coding changes only at codons 20 (D/G) and 225 (S/F). Determination of PRNP functional gene alleles from 47 CWD-positive mule deer showed the predominant allele encoded 20D225S (frequency 0.85). When alleles were grouped by coding changes in the functional gene, four of the six possible peptide combinations were identified in infected deer. Three pseudogene alleles with coding changes in exon 3 were identified in the mule deer samples examined. Because the TSEs appear to be "protein only" disorders, the presence of an untranslated pseudogene is not expected to affect disease resistance. Therefore, selection of a genotyping method specific for the functional gene is critical for large-scale studies to identify the role of the PRNP gene in susceptibility to CWD in mule deer.

Alleles↗

PubMed2006

Requirements for polyadenylation at the 3' end of LINE-1 elements.

LINE-1 (L1) is the only active, autonomous, non-LTR, human retroelement. There are about 5x10(5) L1 copies in the human genome, the majority of which are truncated at their 5' ends. Both truncated and full-length L1 insertions contain a polyadenylation (polyA) signal at their 3' ends. A typical polyA site consists of the three main cis-acting elements: a conserved hexamer, cleavage site, and a GU-rich downstream region. A newly inserted L1 copy contains the conserved AATAAA hexamer at the end of its sequence. However, the GU-rich downstream region has to be provided by the neighboring genomic sequences and therefore it would vary for every L1 copy. Using northern blot analysis of transiently transfected L1 expression vectors we demonstrate that L1 element contain sequence that allow efficient polyadenylation at the L1 3' end upon retrotransposition into a new genomic location independent of the base composition downstream of the insertion site. The strategy of polyadenylation at the 3' end of L1 parallels the approach the element employs at its 5'UTR by having an unusual internal polymerase II promoter, making new insertions less dependent on the properties of the flanking sequences at the new locus.

3' Untranslated Regions↗

PubMed2006

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

Amino Acid Sequence↗

PubMed2006

Selection against LINE-1 retrotransposons results principally from their ability to mediate ectopic recombination.

LINE-1 (L1) retrotransposons constitute the most successful family of autonomous retroelements in mammals and they represent at least 17% of the size of the human genome. L1 insertions have occasionally been recruited to perform a beneficial function but the vast majority of L1 inserts are either neutral or deleterious. The basis for the deleterious effect of L1 remains a matter of debate and three possible mechanisms have been suggested: the direct effect of L1 inserts on gene activity, genetic rearrangements caused by L1-mediated ectopic recombination, or the retrotransposition process per se. We performed a genome-wide analysis of the distribution of L1 retrotransposons relative to the local recombination rate and the age and length of the elements. The proportion of L1 elements that are longer than 1.2 Kb is higher in low-recombining regions of the genome than in regions with a high recombination rate, but the genomic distributions of full-length elements (i.e. elements capable of retrotransposition) and long truncated elements were indistinguishable. We also found that the intensity of selection against long elements is proportional to the replicative success of L1 families. This suggests that the deleterious effect of L1 elements results principally from their ability to mediate ectopic recombination.

Chromosomes, Human↗

PubMed2006

Role of endogenous retroviruses in autoimmune diseases.

Molecular epidemiologic proof that HERVs and other retroelements are involved in autoimmunity or other disorders is complicated by their large numbers in the human genome. As discussed, most HERVs are no longer functional or active because of the accumulation of mutations, frameshifts, and deletions. Detection or quantification of HERV transcripts that may be pathologically involved in a particular autoimmune disease thus is often compromised by the presence in great excess of related, but nonfunctional, RNA. This phenomenon should not deter active work in the field, although it will require development of improved methods to discriminate accurately between closely related RNA transcripts. Development of improved immunologic methods to precisely identify epitopes on autoantigens or rare self-reactive T-cell clones may further implicate HERVs and the other repetitive elements in regulation of the immune system in health and disease.

Animals↗

PubMed2006

Molecular and cellular basis of rheumatoid joint destruction.

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with joint destruction. Synovial fibroblasts are key players in this pathological process. They favorise a pro-inflammatory environment in the synovial tissue, interact with the immune system and regulate the differentiation of monocytes into osteoclasts. Synovial hyperplasia is another characteristic of RA, reflecting not only an imbalance between proliferation and apoptosis, but also the migration of cells into the synovial tissue. Gene transfer experiments have been used as important tools for the understanding of molecular and cellular changes that characterize the activated RA synovial fibroblasts. Activated synovial fibroblasts can invade cartilage and bone. Synovial activation is driven by cytokines, such as TNFalpha and IL-1, as well as IL-15, 16, 17, 18, 22, 23, but also by cytokine-independent mechanisms that involve the innate immune system (i.e. TLRs), a unique communication network of microparticles and epigenetic changes (e.g. L1 retroelements).

Animals↗

PubMed2004

Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes.

Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.

Algorithms↗

PubMed2005

Nickel stimulates L1 retrotransposition by a post-transcriptional mechanism.

Sequence studies of the human genome demonstrate that almost half of the DNA is derived from mobile elements. Most of the current retrotransposition activity arises from L1 and the L1-dependent, non-autonomous elements, such as Alu, contributing to a significant amount of genetic mutation and genomic instability. We present data demonstrating that nickel chloride, but not cobalt chloride, is able to stimulate L1 retrotransposition about 2.5-fold. Our data suggest that the stimulation occurs at a post-transcriptional level, possibly during the integration process. The effect of nickel on the cell is highly complex, limiting the determination of the exact mechanism of this stimulation. The observed stimulation of L1 retrotransposition is not due to a general increase in L1 transcription or an increase in the number of genomic nicks caused by nickel, but more likely caused by a decrease in DNA repair activities that influence the downstream events of retrotransposition. Our observations demonstrate the influence of environmental toxicants on human retroelement activity. We present an additional mechanism for heavy-metal carcinogenesis, where DNA damage through mobile element activation must be considered when dealing with genomic damage/instability in response to environmental agents.

Cell Cycle↗

PubMed2006

The human LINE-1 retrotransposon creates DNA double-strand breaks.

Long interspersed element-1 (L1) is an autonomous retroelement that is active in the human genome. The proposed mechanism of insertion for L1 suggests that cleavage of both strands of genomic DNA is required. We demonstrate that L1 expression leads to a high level of double-strand break (DSB) formation in DNA using immunolocalization of gamma-H2AX foci and the COMET assay. Similar to its role in mediating DSB repair in response to radiation, ATM is required for L1-induced gamma-H2AX foci and for L1 retrotransposition. This is the first characterization of a DNA repair response from expression of a non-long terminal repeat (non-LTR) retrotransposon in mammalian cells as well as the first demonstration that a host DNA repair gene is required for successful integration. Notably, the number of L1-induced DSBs is greater than the predicted numbers of successful insertions, suggesting a significant degree of inefficiency during the integration process. This result suggests that the endonuclease activity of endogenously expressed L1 elements could contribute to DSB formation in germ-line and somatic tissues.

Animals↗