Competition between R1 and R2 transposable elements in the 28S rRNA genes of insects

R1 and R2 are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. R1 elements insert into a site that is 74 bp downstream of the R2 insertion site, thus the presence of an R2 in the same 28S gene may inhibit the expression of R1. Consistent with such a suggestion, the R1 elements of Drosophila melanogaster have a strong bias against inserting into 28S genes already containing an R2 element. R2 elements, on the other hand, are only 2-3 fold inhibited from inserting into a 28S gene already containing an R1. D. melanogaster R1 elements are unusual in that they generate a 23-bp deletion of the target site upstream of the insertion. Using in vitro assays developed to study R2 integration, we show that the presence of R1 sequences 51 bp downstream of the R2 insertion site changes the nucleosomal structure that can be formed by the R2 target site. The R2 endonuclease is inhibited from cleaving these altered nucleosomes. We suggest that R1 elements have been selected to make this large deletion of the 28S gene to block the insertion of an upstream R2 element. These findings are consistent with the model that R1 and R2 are in competition for the limited number of insertion sites available within their host's genome.     

Evolution of target specificity in R1 clade non-LTR retrotransposons

Although most non-long terminal repeat (non-LTR) retrotransposons are inserted throughout the host genome, many non-LTR elements in the R1 clade are inserted into specific sites within the target sequence. Four R1 clade families have distinct target specificity: R1 and RT insert into specific sites of 28S rDNA, and TRAS and SART insert into different sites within the (TTAGG)(n) telomeric repeats. To study the evolutionary history of target specificity of R1-clade retrotransposons, we have screened extensively novel representatives of the clade from various insects by in silico and degenerate polymerase chain reaction (PCR) cloning. We found four novel sequence-specific elements; Waldo (WaldoAg1, 2, and WaldoFs1) inserts into ACAY repeats, Mino (MinoAg1) into AC repeats, R6 into another specific site of the 28S rDNA, and R7 into a specific site of the 18S rDNA. In contrast, several elements (HOPE, WISHBm1, HidaAg1, NotoAg1, KagaAg1, Ha1Fs1) lost target sequence specificity, although some of them have preferred target sequences. Phylogenetic trees based on the RT and EN domains of each element showed that (1) three rDNA-specific elements, RT, R6, and R7, diverged from Waldo; (2) the elements having similar target sequences are phylogenetically related; and (3) the target specificity in the R1 clade was obtained once and thereafter altered and lost several times independently. These data indicate that the target specificity in R1 clade retroelements has changed during evolution and is more divergent than has been speculated so far.     

Rates of R1 and R2 retrotransposition and elimination from the rDNA locus of Drosophila melanogaster

R1 and R2 elements are non-LTR retrotransposons that insert specifically into the 28S rRNA genes of arthropods. The process of concerted evolution of the rDNA locus should give rise to rapid turnover of these mobile elements compared to elements that insert at sites throughout a genome. To estimate the rate of R1 and R2 turnover we have examined the insertion of new elements and elimination of old elements in the Harwich mutation accumulation lines of Drosophila melanogaster, a set of inbred lines maintained for >350 generations. Nearly 300 new insertion and elimination events were observed in the 19 Harwich lines. The retrotransposition rate for R1 was 18 times higher than the retrotransposition rate for R2. Both rates were within the range previously found for retrotransposons that insert outside the rDNA loci in D. melanogaster. The elimination rates of R1 and R2 from the rDNA locus were similar to each other but over two orders of magnitude higher than that found for other retrotransposons. The high rates of R1 and R2 elimination from the rDNA locus confirm that these elements must maintain relatively high rates of retrotransposition to ensure their continued presence in this locus.     

NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.     

R1 and R2 retrotransposition and deletion in the rDNA loci on the X and Y chromosomes of Drosophila melanogaster

The non-LTR retrotransposons R1 and R2 insert into the 28S rRNA genes of arthropods. Comparisons among Drosophila lineages have shown that these elements are vertically inherited, while studies within species have indicated a rapid turnover of individual copies (elimination of old copies and the insertion of new copies). To better understand the turnover of R1 and R2, 200 retrotranspositions and nearly 100 eliminations have been scored in the Harwich mutation-accumulation lines of Drosophila melanogaster. Because the rDNA arrays in D. melanogaster are present on the X and Y chromosomes and no exchanges were detected in these lines, it was possible to show that R1 retrotranspositions occur predominantly in the male germ line, while R2 retrotranspositions were more evenly divided between the germ lines of both sexes. The rate of elimination of elements from the Y rDNA array was twice that of the X rDNA array with both chromosomal loci containing regions where the rate of elimination was on average eight times higher. Most R1 and R2 eliminations appear to occur by large intrachromosomal events (i.e., loop-out events) that involve multiple rDNA units. These findings are interpreted in light of the known abundance of R1 and R2 elements in the X and Y rDNA loci of D. melanogaster.     

Identification of rDNA-specific non-LTR retrotransposons in Cnidaria

Ribosomal RNA genes are abundant repetitive sequences in most eukaryotes. Ribosomal DNA (rDNA) contains many insertions derived from mobile elements including non-long terminal repeat (non-LTR) retrotransposons. R2 is the well-characterized 28S rDNA-specific non-LTR retrotransposon family that is distributed over at least 4 bilaterian phyla. R2 is a large family sharing the same insertion specificity and classified into 4 clades (R2-A, -B, -C, and -D) based on the N-terminal domain structure and the phylogeny. There is no observation of horizontal transfer of R2; therefore, the origin of R2 dates back to before the split between protostomes and deuterostomes. Here, we in silico identified 1 R2 element from the sea anemone Nematostella vectensis and 2 R2-like retrotransposons from the hydrozoan Hydra magnipapillata. R2 from N. vectensis was inserted into the 28S rDNA like other R2, but the R2-like elements from H. magnipapillata were inserted into the specific sequence in the highly conserved region of the 18S rDNA. We designated the Hydra R2-like elements R8. R8 is inserted at 37 bp upstream from R7, another 18S rDNA-specific retrotransposon family. There is no obvious sequence similarity between targets of R2 and R8, probably because they recognize long DNA sequences. Domain structure and phylogeny indicate that R2 from N. vectensis is the member of the R2-D clade, and R8 from H. magnipapillata belongs to the R2-A clade despite its different sequence specificity. These results suggest that R2 had been generated before the split between cnidarians and bilaterians and that R8 is a retrotransposon family that changed its target from the 28S rDNA to the 18S rDNA.     

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.     

Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm

A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2. These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner. We report here the functional expression in E. coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site. The resulting cleavage is a 4 bp staggered 5' overhang. Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site. A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.     

Site-specific ribosomal DNA insertion elements in Anopheles gambiae and A. arabiensis: nucleotide sequence of gene-element boundaries.

The nucleotide sequence of the junctions between the 28S ribosomal gene and site-specific insertion elements from two sibling mosquito species, Anopheles gambiae and A. arabiensis, is reported. In both species, elements insert at the same point within the 28S gene, but this site is 634 basepairs (bp) 3' of the R1 (Type I) insertion site in Drosophila melanogaster. The two mosquito elements each have poly A tails and a polyadenylation signal, but the extreme 3' and 5' ends show no other similarity to each other or to any other insertion element. In both mosquito species, identical target site duplications of 17 bp are generated. The sequence TNTCCCTNT found in this duplication is also found in the 14 bp target site duplications that flank R1 elements in D. melanogaster. Another sequence in this duplication, GGGATAACT, is very similar to the sequence GGGAGTAACT found in the 24 base sequence required by the Bombyx mori R2 endonuclease.