non-LTR Retrotransposable Elements Research Articles

Generation of microsatellite repeat families by RTE retrotransposons in lepidopteran genomes

BackgroundDeveloping lepidopteran microsatellite DNA markers can be problematical, as markers often exhibit multiple banding patterns and high frequencies of non-amplifying "null" alleles. Previous studies identified sequences flanking simple sequence repeat (SSR) units that are shared among many lepidopteran species and can be grouped into microsatellite-associated DNA families. These families are thought to be associated with unequal crossing-over during DNA recombination or with transposable elements (TEs).ResultsWe identified full-length lepidopteran non-LTR retrotransposable elements of the RTE clade in Heliconius melpomene and Bombyx mori. These retroelements possess a single open reading frame encoding the Exonuclease/Endonuclease/Phosphatase and the Reverse Transcriptase/nLTR domains, a 5' UTR (untranslated region), and an extremely short 3' UTR that regularly consists of SSR units. Phylogenetic analysis supported previous suggestions of horizontal transfer among unrelated groups of organisms, but the diversity of lepidopteran RTE elements appears due to ancient divergence of ancestral elements rather than introgression by horizontal transfer. Similarity searches of lepidopteran genomic sequences in GenBank identified partial RTE elements, usually consisting of the 3' terminal region, in 29 species. Furthermore, we identified the C-terminal end of the Reverse Transcriptase/nLTR domain and the associated 3' UTR in over 190 microsatellite markers from 22 lepidopteran species, accounting for 10% of the lepidopteran microsatellites in GenBank. Occasional retrotransposition of autonomous elements, frequent retrotransposition of 3' partial elements, and DNA replication slippage during retrotransposition offers a mechanistic explanation for the association of SSRs with RTE elements in lepidopteran genomes.ConclusionsNon-LTR retrotransposable elements of the RTE clade therefore join a diverse group of TEs as progenitors of SSR units in various organisms. When microsatellites are isolated using standard SSR enrichment protocols and primers designed at complementary repeated regions, amplification from multiple genomic sites can cause scoring difficulties that compromise their utility as markers. Screening against RTE elements in the isolation procedure provides one strategy for minimizing this problem.

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion

Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

RetroPred: A tool for prediction, classification and extraction of non-LTR retrotransposons (LINEs & SINEs) from the genome by integrating PALS, PILER, MEME and ANN.

The problem of predicting non-long terminal repeats (LTR) like long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) from the DNA sequence is still an open problem in bioinformatics. To elevate the quality of annotations of LINES and SINEs an automated tool "RetroPred" was developed. The pipeline allowed rapid and thorough annotation of non-LTR retrotransposons. The non-LTR retrotransposable elements were initially predicted by Pairwise Aligner for Long Sequences (PALS) and Parsimonious Inference of a Library of Elementary Repeats (PILER). Predicted non-LTR elements were automatically classified into LINEs and SINEs using ANN based on the position specific probability matrix (PSPM) generated by Multiple EM for Motif Elicitation (MEME). The ANN model revealed a superior model (accuracy = 78.79 +/- 6.86 %, Q(pred) = 74.734 +/- 17.08 %, sensitivity = 84.48 +/- 6.73 %, specificity = 77.13 +/- 13.39 %) using four-fold cross validation. As proof of principle, we have thoroughly annotated the location of LINEs and SINEs in rice and Arabidopsis genome using the tool and is proved to be very useful with good accuracy. Our tool is accessible at http://www.juit.ac.in/RepeatPred/home.html.

Characterization of Active R2 Retrotransposition in the rDNA Locus of Drosophila simulans

The rRNA gene (rDNA) loci of all arthropod lineages contain non-LTR retrotransposable elements that have evolved to specifically insert into the 28S rRNA genes. Extensive in vitro experiments have been conducted to investigate the mechanism of R2 retrotransposition but little is known of the insertion frequency or cellular factors that might regulate R2 activity. In this article, isofemale lines obtained from a population of Drosophila simulans were surveyed for recent R2 insertions. Within most lines, all individuals showed the same collection of R2 insertions, providing no evidence for recent R2 activity. However, in a few of the isofemale lines, virtually all individuals differed in their R2 insertion profiles. The descendants of individual pairs of flies from these "active lines" rapidly accumulated new insertions. The frequent insertion of new R2 elements was associated with the elimination of old R2 elements from the rDNA locus. The existence of lines in which R2 retrotransposes frequently and lines in which the elements appear dormant suggests that cellular mechanisms that can regulate the activity of R2 exist. Retrotransposition activity was correlated with the number of full-length R2 elements but not with the size of the rDNA locus or the number of uninserted units.

Gene organization of bovine BCNT that contains a portion corresponding to an endonuclease domain derived from an RTE-1 (Bov-B LINE), non-LTR retrotransposable element: duplication of an intramolecular repeat unit downstream of the truncated RTE-1

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion. The 3′ UTR of bovine bcnt cDNA also contains an approximately 300-bp portion homologous to the 3′-part of RTE-1. We examined the bovine bcnt genomic DNA sequence to understand how the bovine bcnt gene has been organized. The sequence of 3′ UTR homologous portion was found to more closely resemble the Art2 element than the bovine RTE-1. By PCR screening a bovine/hamster hybrid somatic cell panel, the bovine bcnt gene was mapped to chromosome 18, syntenic human chromosome 16q on which human BCNT is located. The bcnt genomic DNA sequence corresponding to the cDNA downstream of a RTE-1 derived portion reveals that each IR unit is flanked by both 5′-side and 3′-side introns and that 3′-UTR consists of one exon. The alignment of the above sequence with a bovine RTE-1 did not show any significant homology downstream of the endonuclease domain. On the other hand, the alignment of the intron sequences with each other revealed that the six sequential homologous segments ranging in size from 40 to 453 bp existed over a 1 kb long sequence between both the 5′- and 3′-side introns flanking each bovine IR unit. In addition, both the 174-bp of 5′-side intron and 80-bp of 3′-side intron neighboring each 120-bp IR exon are significantly homologous among the two bovine IRs, human IR and mouse IR. These results suggest that a truncated bovine RTE-1 was inserted into the intron upstream of an IR unit of an ancestor bcnt gene and that a duplication of a relatively long region that includes IR occurred in the bovine genome.

Ikirara insertions reveal five new Anopheles gambiae transposable elements in islands of repetitious sequence.

Characterization of Anopheles gambiae genomic clones containing Ikirara inverted repeats revealed five novel sequences related to known transposable elements (TEs). One TE is related to the mariner/Tc1 superfamily of class II (DNA-to-DNA) transposons, while four are related to class I (RNA-mediated transposition) elements. Crusoe, the class II element; is most similar to the Caenorhabditis elegans transposon Tc1-like TEs. Vash elements, represented twice in our clones, are related to the Q/T1 family of A. gambiae non-LTR retrotransposable elements. Guildenstern is a member of the RT1 and RT2 non-LTR retrotransposon family. Although RT1 and RT2 elements normally have a highly stereotyped insertion preference for sequences within ribosomal genes, Guildenstern is not located in ribosomal sequence. JuanAg is the first anopheline member of the mosquito non-LTR retrotransposon family of Juan elements that previously had included just the culicine elements JuanA and JuanC. Approximately 753 bp is missing from the central portion of the JuanAg reverse transcriptase gene, where an Ikirara inverted repeat is found in its stead. Ozymandias, the only LTR retrotransposon found in the clones, is most similar to the Drosophila melanogaster 412 element. Single Ikirara inverted repeats were also found adjacent to nontransposable element repetitious sequences. Our analysis suggests that the A. gambiae genome organization could best be described as islands of short-period interspersion repetitious DNA in a sea of long-period interspersion, mostly unique sequence DNA.

Conserved features at the 5 end of Drosophila R2 retrotransposable elements: implications for transcription and translation.

R2 non-LTR retrotransposable elements insert site-specifically into the 28S ribosomal genes of insects. The sequence of the 5' end of full-length R2 elements from thirteen species of Drosophila were compared. Sequences within the 5' untranslated region (5' UTR) revealed little to suggest the presence of a promoter. Protein translation initiates within the 5' UTR and requires the bypassing of a highly conserved termination codon preceding the single R2 open reading frame. This bypassing probably involves a conserved RNA secondary structure which brings a potential initiation codon into close proximity to this termination codon. The most highly conserved sequence within the 5' UTR has properties similar to internal ribosomal entry sites. Based on these findings, we propose that R2elements are co-transcribed with the 28S gene and are translated as part of a large ribosomal subunit.

Genome size as a mutation-selection-drift process.

A novel method for estimating neutral rates and patterns of DNA evolution in Drosophila takes advantage of the propensity of non-LTR retrotransposable elements to create nonfunctional, transpositionally inactive copies as a product of transposition. For many LINE elements, most copies present in a genome at any one time are nonfunctional "dead-on-arrival" (DOA) copies. Because these are off-shoots of active, transpositionally competent "master" lineages, in a gene tree of a LINE element from multiple samples from related species, the DOA lineages are expected to map to the terminal branches and the active lineages to the internal branches, the primary exceptions being when the sample includes DOA copies that are allelic or orthologous. Analysis of nucleotide substitutions and other changes along the terminal branches therefore allows estimation of the fixation process in the DOA copies, which are unconstrained with respect to protein coding; and under selective neutrality, the fixation process estimates the underlying mutational pattern. We have studied the retroelement Helena in Drosophila. An unexpectedly high rate of DNA loss was observed, yielding a half-life of unconstrained DNA sequences approximately 60-fold faster in Drosophila than in mammals. The high rate of DNA loss suggests a straightforward explanation of the seeming paradox that Drosophila has many fewer pseudogenes than found in mammalian species. Differential rates of deletion in different taxa might also contribute to the celebrated C-value paradox of why some closely related organisms can have very different DNA contents. New data presented here rule out the possibility that the transposition process itself is highly mutagenic, hence the observed linear relation between number of deletions and number of nucleotide substitutions is most easily explained by the hypothesis that both types of changes accumulate in unconstrained sequences over time.

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups.

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.

Trash DNA is what gets thrown away: high rate of DNA loss in Drosophila

We have recently described a novel method of estimating neutral rates and patterns of spontaneous mutation (Petrov et al., 1996). This method takes advantage of the propensity of non-LTR retrotransposable elements to create non-functional, ‘dead-on-arrival’ copies as a product of transposition. Maximum parsimony analysis is used to separate the evolution of actively transposing lineages of a non-LTR element from the fate of individual inactive insertions, and thereby allows one to assess directly the relative rates of different types of mutation, including point substitutions, deletions and insertions. Because non-LTR elements enjoy wide phylogenetic distribution, this method can be used in taxa that do not harbor a significant number of bona fide pseudogenes, as is the case in Drosophila (Jeffs and Ashburner, 1991; Weiner et al., 1986). We used this method with Helena, a non-LTR retrotransposable element present in the Drosophila virilis species group. A striking finding was the virtual absence of insertions and remarkably high incidence of large deletions, which combine to produce a high overall rate of DNA loss. On average, the rate of DNA loss in D. virilis is approximately 75 times faster than that estimated for mammalian pseudogenes (Petrov et al., 1996). The high rate of DNA loss should lead to rapid elimination of non-essential DNA and thus may explain the seemingly paradoxical dearth of pseudogenes in Drosophila. Varying rates of DNA loss may also contribute to differences in genome size (Graur et al., 1989; Petrov et al., 1996), thus explaining the celebrated ‘C-value’ paradox (John and Miklos, 1988). In this paper we outline the theoretical basis of our method, examine the data from this perspective, and discuss potential problems that may bias our estimates.

Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: A mechanism for non-LTR retrotransposition

R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3′ hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.