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tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Binding Sites↗

A novel insertion of a rearranged L1 element in exon 44 of the dystrophin gene: further evidence for possible bias in retroposon integration.

L1 elements are mammalian retrotransposons contributing to genome evolution and causing rare mutations in human. We describe a de novo insertion of an L1 element into the dystrophin gene resulting in skipping of exon 44 and causing Duchenne muscular dystrophy in a boy. The L1 element was rearranged due to the twin-priming mechanism, but contrary to all described L1 rearrangements the 5' region of the inverted L1 sequence ended within the poly(A) tail of the element. Furthermore, the target site for the insertion was located only 87 bp from the insertion site in another patient described previously. These findings can contribute to the understanding of the mechanisms of L1 element rearrangement, and may support the notion that some subregions of the human genome could be preferred targets for retroelements using the L1 enzymatic machinery.

Base Sequence↗

Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases.

The mammalian APOBEC3 family of cytidine deaminases includes several members that possess potent antiretroviral activity. Human APOBEC3F and APOBEC3G are specifically incorporated into human immunodeficiency virus type 1 (HIV-1) progeny virions in the absence of virion infectivity factor (Vif), where they deaminate deoxycytidine to deoxyuridine on the minus strand of nascent reverse transcripts. Editing of the HIV-1 cDNA leads to its degradation or to G to A hypermutation of the integrated provirus. Here, we show that APOBEC3 proteins also restrict the activity of a distantly related long terminal repeat (LTR) retrotransposon. When expressed in the yeast Saccharomyces cerevisiae, human APOBEC3C, APOBEC3F, or APOBEC3G or mouse APOBEC3 potently inhibit replication of the Ty1 LTR retrotransposon. APOBEC3G interacts with Ty1 Gag and is packaged into Ty1 virus-like particles (VLPs) by a mechanism that closely resembles the one it uses to enter HIV-1 virions. Expression of APOBEC3G results in a reduced level of Ty1 cDNA integration and G to A editing of integrated Ty1 cDNA. Our findings indicate that APOBEC3G restricts Ty1 and HIV-1 by similar mechanisms and suggest that the APOBEC3 proteins target a substantially broader spectrum of retroelements than previously appreciated.

APOBEC-3G Deaminase↗

Global heterochromatic colocalization of transposable elements with minisatellites in the compact genome of the pufferfish Tetraodon nigroviridis.

Because of its unusual high degree of compaction and paucity of repetitive sequences, the genome of the smooth pufferfish Tetraodon nigroviridis is the subject of a well-advanced sequencing project. An astonishing diversity of transposable elements not found in the human and the mouse has been observed in the genome of T. nigroviridis. Due to the difficulty of assembling repeat-rich regions, the whole genome shotgun sequencing approach will probably fail to reveal the general organisation of this compact vertebrate genome. Therefore, in order to gain new insights into the global distribution pattern of repeated DNA in the genome of T. nigroviridis, we have reconstructed partial/complete repetitive sequences from data generated by the genome project and performed double-colour fluorescent in situ hybridization (FISH) analysis for representatives of three major categories of repeated sequences including two minisatellites (ms100 and ms104), two DNA transposons (Tol2 and Buffy1) and two non-long terminal repeat (LTR) retrotransposons (Rex3 and Babar). We show that DNA transposons and retroelements very frequently colocalize with minisatellites and mostly accumulate within heterochromatic regions. These results, which have not been reported so far for the fugu Takifugu rubripes, show that repeated elements are generally excluded from gene-rich regions in T. nigroviridis and underline the extreme degree of compartmentalization of this compact genome. The genome organization of the pufferfish is clearly different from that observed in humans, where repeated sequences make up an important fraction of euchromatic DNA, and is more similar to that observed in the fruit fly Drosophila melanogaster.

Amino Acid Sequence↗

Two families of non-LTR retrotransposons, Syrinx and Daphne, from the Darwinulid ostracod, Darwinula stevensoni.

Two novel families of non-LTR retrotransposons, named Syrinx and Daphne, were cloned and characterized in a putative ancient asexual ostracod Darwinula stevensoni. Phylogenetic analysis reveals that Daphne is the founding member of a novel clade of non-LTR retroelements, which also contains retrotransposon families from the sea urchin and the silkworm and forms a sister clade to L2-like elements. The Syrinx family of non-LTR retrotransposons exhibits evidence of relatively recent activity, manifested in high levels of sequence similarity between individual copies and a three- to ten-fold excess of synonymous substitutions, which is indicative of purifying selection. The Daphne family may have very few copies with intact open reading frames, and exhibits neutral within-family ratio of non-synonymous to synonymous substitutions. It can additionally be characterized by formation of inverted truncated head-to-head structures. All of these features make recent activity less likely than in the Syrinx family. Our results are discussed in light of the evolutionary consequences of long-term asexuality in general and in D. stevensoni in particular.

Amino Acid Sequence↗

Life without GAG: the BARE-2 retrotransposon as a parasite's parasite.

A large proportion of the plant LTR (Long Terminal Repeat) retrotransposons are partly or completely unable to synthesize their own machinery for transposition. However, most of these inactive or non-autonomous elements are likely able to retrotranspose, based on their insertional polymorphism. Therefore, they must be parasitic on one or more active partners. Here, we describe the parasitism of the chimeric BARE-2 element on the active BARE-1 (Barley RetroElement-2 and -1 respectively). These two elements are present in the Triticeae and related species, and are together polymorphic among closely related accessions. BARE-2 elements are unable to synthesize their own GAG protein, and harbor a specific ATG deletion in the gag ORF. However, BARE-2 sequences are conserved with BARE-1 in the PBS (Primer Binding Site), PSI (Packaging SIgnal) and DIS (DImerization Signal) domains. As these motifs have been shown to allow parasitism among the lentiviruses, we conclude that BARE-2 is probably a partial parasite of the BARE-1 element because the machinery of the latter can complement the defective GAG of the former. This example emphasizes that we must characterize the parasitic network of LTR retrotransposons and its implication for integration of autonomous, inactive, and non-autonomous elements in order to understand current and past host genome evolution.

Base Sequence↗

A deep-branching clade of retrovirus-like retrotransposons in bdelloid rotifers.

Rotifers of class Bdelloidea, a group of aquatic invertebrates in which males and meiosis have never been documented, are also unusual in their lack of multicopy LINE-like and gypsy-like retrotransposons, groups inhabiting the genomes of nearly all other metazoans. Bdelloids do contain numerous DNA transposons, both intact and decayed, and domesticated Penelope-like retroelements Athena, concentrated at telomeric regions. Here we describe two LTR retrotransposons, each found at low copy number in a different bdelloid species, which define a clade different from previously known clades of LTR retrotransposons. Like bdelloid DNA transposons and Athena, these elements are found preferentially in telomeric regions. Unlike bdelloid DNA transposons, many of which are decayed, the newly described elements, named Vesta and Juno, inhabiting the genomes of Philodina roseola and Adineta vaga, respectively, appear to be intact and represent recent insertions, possibly from an exogenous source. We describe the retrovirus-like structure of the new elements, containing gag, pol, and env-like open reading frames, and discuss their possible origins, transmission, and behavior in bdelloid genomes.

Amino Acid Sequence↗

Survey of long terminal repeat retrotransposons of domesticated silkworm (Bombyx mori).

Long terminal retrotransposons are major components of eukaryotic transposable elements. We have surveyed the long terminal repeats (LTR) retrotransposons of domesticated silkworm (Bombyx mori) by mining the data produced by Bombyx mori Genome Sequencing Project. At least 29 separate families of LTR retrotransposons are identified in this survey, comprising of 11.8% of the complete sequence. Families of domesticated silkworm LTR retrotransposons can be mainly classified into three groups: gypsy-like, copia-like, Pao-Bel. Fourteen families identified consist of gypsy-like elements, four families consist of copia-like elements and seven families consist of Pao-Bel elements. In addition to the three groups of LTR retrotransposons, two families of unusual non-coding elements are identified in the genome of this species. Further phylogenetic analysis of RT domain indicates that the elements of B.mori show high diversity and can form different clades in each group. An analysis of sequence variation from different families reveals distinct patterns of variation for the elements belonging to three groups. The analysis of the domesticated silkworm LTR retrotransposons should assist in our understanding of the roles of retroelement in lepidopteron insect genome evolution.

Animals↗

The varying microsporidian genome: existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis.

Microsporidia are a group of intracellular parasites with an extremely compact genome and there is no confirmed evidence that retroelements are parasitised in these organisms. Using the dataset of 200,000 genomic shotgun reads of the silkworm pebrine Nosema bombycis, we have identified the eight complete N. bombycis long-terminal repeat retrotransposon (Nbr) elements. All of the Nbr elements are Ty3/gypsy members and have close relationships to Saccharomycetes long-terminal repeat retrotransposons identified previously, providing further evidence of their relationship to fungi. To explore the effect of retrotransposons in microsporidian genome evolution, their distribution was characterised by comparisons between two N. bombycis contigs containing the Nbr elements with the completed genome of the human parasite Encephalitozoon cuniculi, which is closely related to N. bombycis. The Nbr elements locate between or beside syntenic blocks, which are often clustered with other transposable-like sequences, indicating that they are associated with genome size variation and syntenic discontinuities. The ratios of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site of the open reading frames among members of each of the eight Nbr families were estimated, which reveal the purifying selection acted on the N. bombycis long-terminal repeat retrotransposons. These results strongly suggest that retrotransposons play a major role in reorganization of the microsporidian genome and they might be active. The present study presents an initial characterization of some transposable elements in the N. bombycis genome and provides some insight into the evolutionary mechanism of microsporidian genomes.

Amino Acid Sequence↗

Evolution of non-LTR retrotransposons in the trypanosomatid genomes: Leishmania major has lost the active elements.

The ingi and L1Tc non-LTR retrotransposons--which constitute the ingi clade--are abundant in the genome of the trypanosomatid species Trypanosoma brucei and Trypanosoma cruzi, respectively. The corresponding retroelements, however, are not present in the genome of a closely related trypanosomatid, Leishmania major. To study the evolution of non-LTR retrotransposons in trypanosomatids, we have analyzed all ingi/L1Tc elements and highly degenerate ingi/L1Tc-related sequences identified in the recently completed T. brucei, T. cruzi and L. major genomes. The coding sequences of 242 degenerate ingi/L1Tc-related elements (DIREs) in all three genomes were reconstituted by removing the numerous frame shifts. Three independent phylogenetic analyses conducted on the conserved domains encoded by these elements show that all DIREs, including the 52 L. major DIREs, form a monophyletic group belonging to the ingi clade. This indicates that the trypanosomatid ancestor contained active mobile elements that have been retained in the Trypanosoma species, but were lost from L. major genome, where only remnants (DIRE) are detectable. All 242 DIREs analyzed group together according to their species origin with the exception of 11 T. cruzi DIREs which are close to the T. brucei ingi/DIRE families. Considering the absence of known horizontal transfer between the African T. brucei and the South-American T. cruzi, this suggests that this group of elements evolved at a lower rate when compared to the other trypanosomatid elements. Interestingly, the only nucleotide sequence conserved between ingi and L1Tc (the first 79 residues) is also present at the 5'-extremity of all the full length DIREs and suggests a possible role for this conserved motif, as well as for DIREs.

Animals↗

Reverse transcriptase and reverse splicing activities encoded by the mobile group II intron cobI1 of fission yeast mitochondrial DNA.

Mobile group II introns encode multidomain proteins with maturase activity involved in splicing and reverse transcriptase (RT) and (often) endonuclease activities involved in intron mobility. These activities are present in a ribonucleoprotein complex that contains the excised intron RNA and the intron-encoded protein. Here, we report biochemical studies of the protein encoded by the group IIA1 intron in the cob gene of fission yeast Schizosaccharomyces pombe mitochondria (cobI1). RNP particle fractions from the wild-type fission yeast strain with cobI1 in its mtDNA have RT activity even without adding an exogenous primer. Characterization of the cDNA products of such reactions showed a strong preference for excised intron RNA as template. Two main regions for initiation of cDNA synthesis were mapped within the intron, one near the DIVa putative high-affinity binding site for the intron-encoded protein and the other near domain VI. Adding exogenous primers complementary to cob exon 2 sequences near the intron/exon boundary stimulated RT activity but mainly for pre-mRNA rather than mRNA templates. Further in vitro experiments demonstrated that cobI1 RNA in RNP particle fractions can reverse splice into double-stranded DNA substrates containing the intron homing site. Target DNA primed reverse transcription was not detected unless a DNA target was used that was already nicked in the antisense strand of exon 2. This study shows that S.pombe cobI1 encodes RNP particles that have most of the biochemical activities needed for it to be a retroelement. Interestingly, it appears to lack an endonuclease activity, suggesting that the active homing exhibited by this intron in crosses may differ somewhat from that of the better-characterized introns.

Blotting, Northern↗

Targeting survival: integration site selection by retroviruses and LTR-retrotransposons.

Replication of retroviruses and retrotransposons depends on selecting a favorable chromosomal site for integration of their genomic DNA. Different retroelements meet this challenge by targeting distinctive chromosomal regions. Despite these differences, recent data hints at a common targeting mechanism-tethering of integration complexes to proteins bound at favorable sites.

Animals↗

Structural analysis of the plasmid pAAT56 of the filamentous fungus Alternaria alternata.

The circular DNA plasmid, designated pAAT56, has been isolated from strain T88-56 of the Japanese pear pathotype of Alternaria alternata. We determined the complete nucleotide sequence (5354 bp) of pAAT56 and mapped its possible open reading frames (ORFs). Three long ORFs, ORF1 (1290 bp), ORF2 (1653 bp) and ORF3 (690 bp), and four smaller ORFs, ORF4 to ORF7 (> or = 300 bp), were predicted from the sequence. The potential peptides derived from the ORFs other than ORF2 show no homology to other known proteins from a database search. However, ORF2 has significant homology to the pol gene of retrotransposons. The polypeptide derived from ORF2 includes sequences homologous to the reverse transcriptase (RT) and ribonuclease H (RNase H) domains of the retrotransposon Pol peptide. Phylogenetic comparison of RT domains from the retroelements placed pAAT56 in the Ty3/gypsy group of long terminal repeat (LTR) retrotransposons, most closely linked with those of filamentous fungi. The PCR primers were designed on the basis of nucleotide sequences encoding the highly-conserved amino-acid sequences in RT domains among pAAT56 and fungal retrotransposons. The PCR amplified the DNA fragments that possibly encode RT from strains of filamentous fungi that have been reported to carry retrotransposons. These results suggest that pAAT56 has acquired the pol gene from a Ty3/gypsy-group retrotransposon.

Alternaria↗

A retrotransposon family from the pufferfish (fugu) Fugu rubripes.

In this study we describe the isolation and characterisation of the first full-length vertebrate retrotransposon. Knowledge of vertebrate gypsy LTR-retrotransposons has been limited to short internal sequences from three fish and a corrupt sequence from a salamander. This paper describes the sequence of a full-length (5.645 kb) retrotransposon from the fugu fish Fugu rubripes. The retrotransposon, termed sushi-ichi (032H04), is a representative of a retrotransposon family (sushi) found as multiple copies within the fish genome. Two long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences homologous to protease, reverse transcriptase/RNase H and integrase domains, in that order. Sequence comparisons of the predicted ORFs indicate that this element is related to the gypsy class of LTR-retrotransposons. Specifically, the sushi retrotransposons are most closely related to the retrotransposon group which includes the MAGGY retroelement from the rice blast fungus Magnaporthe grisea and the CfT-1 element from the fungal tomato pathogen Cladosporium fulvum.

Amino Acid Sequence↗

Gypsy-class retrotransposon sequences in organisms related to the leaf mould fungus, Cladosporium fulvum.

Gypsy retrotransposons are a major branch of retroid elements and have been found in a wide diversity of eukaryotic organisms including CfT-1 in the tomato leaf mould pathogen, the fungus Cladosporium fulvum (syn. Fulvia fulva). We have examined organisms that are either ecologically or phylogenetically related to C. fulvum, for elements related to CfT-1. Using PCR and Southern hybridisation, similar sequences were found only in the co-genic fungus Cladosporium cladosporioides. This finding confirms the apparent ubiquity of retroelements, suggests that the phylogeny of retrotransposons is consistent with the phylogeny of their hosts and argues against frequent horizontal gene transfer.

Amino Acid Sequence↗

Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases.

Catalytic RNAs are often regarded as molecular fossils from the RNA World, yet it is usually difficult to get more specific information about their evolution. Here we have investigated the coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases (RTs). Unlike group I introns, there has been no obvious reshuffling between intron RNA structures and ORFs. Of the six classes of intron structures that encode ORFs, three are conventional forms of group II A1, B1, and B2 secondary structures, whereas the remaining classes are bacterial, are possibly associated with the most primitive ORFs, and have unusual features and hybrid features of group IIA and group IIB intron structures. Based on these data, we propose a new model for the evolution of group II introns, designated the retroelement ancestor hypothesis, which predicts that the major RNA structural forms of group II introns developed through coevolution with the intron-encoded protein rather than as independent catalytic RNAs, and that most ORF-less introns are derivatives of ORF-containing introns. The model is supported by the distribution of ORF-containing and ORF-less introns, and by numerous examples of ORF-less introns that contain ORF remnants.

Chloroplasts↗

The dispersal of five group II introns among natural populations of Escherichia coli.

Group II introns are self-splicing RNAs that also act as retroelements in bacteria, mitochondria, and chloroplasts. Group II introns were identified in Escherichia coli in 1994, but have not been characterized since, and, instead, other bacterial group II introns have been studied for splicing and mobility properties. Despite their apparent intractability, at least five distinct group II introns exist naturally in E. coli strains. To illuminate their function and learn how the introns have dispersed in their natural host, we have investigated their distribution in the ECOR reference collection. Two introns were cloned and sequenced to complete their partial sequences. Unexpectedly, southern blots showed all ECOR strains to contain fragments and/or full-length copies of group II introns, with some strains containing up to 15 intron copies. One intron, E.c.14, has two natural homing sites in IS629 and IS911 elements, and the intron can be present in one, both, or neither homing site in a given strain. Nearly all strains that contain full-length introns also contain unfilled homing sites, suggesting either that mobility is highly inefficient or that most full-length copies are nonfunctional. The data indicate independent mobility of the introns, as well as mobility via the host DNA elements, and overall, the pattern of intron distribution resembles that of IS elements.

Base Sequence↗

Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure.

L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome.

Base Sequence↗