Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.     

HeT-A_pi1, a piRNA target sequence in the Drosophila telomeric retrotransposon HeT-A, is extremely conserved across copies and species

The maintenance of the telomeres in Drosophila species depends on the transposition of the non-LTR retrotransposons HeT-A, TAHRE and TART. HeT-A and TART elements have been found in all studied species of Drosophila suggesting that their function has been maintained for more than 60 million years. Of the three elements, HeT-A is by far the main component of D. melanogaster telomeres and, unexpectedly for an element with an essential role in telomere elongation, the conservation of the nucleotide sequence of HeT-A is very low. In order to better understand the function of this telomeric retrotransposon, we studied the degree of conservation along HeT-A copies. We identified a small sequence within the 3' UTR of the element that is extremely conserved among copies of the element both, within D. melanogaster and related species from the melanogaster group. The sequence corresponds to a piRNA target in D. melanogaster that we named HeT-A_pi1. Comparison with piRNA target sequences from other Drosophila retrotransposons showed that HeT-A_pi1 is the piRNA target in the Drosophila genome with the highest degree of conservation among species from the melanogaster group. The high conservation of this piRNA target in contrast with the surrounding sequence, suggests an important function of the HeT-A_pi1 sequence in the co-evolution of the HeT-A retrotransposon and the Drosophila genome.     

Heterochromatic distribution of HeT-A- and TART-like sequences in several Drosophila species

Drosophila melanogaster telomeres contain arrays of two non-LTR retrotransposons called HeT-A and TART. Previous studies have shown that HeT-A- and TART-like sequences are also located at non-telomeric sites in the Y chromosome heterochromatin. By in situ hybridization experiments, we mapped TART sequences in the h16 region of the long arm close to the centromere of the Y chromosome of D. melanogaster. HeT-A sequences were localized in two different regions on the Y chromosome, one very close to the centromere in the short arm (h18-h19) and the other in the long arm (h13-h14). To assess a possible heterochromatic location of TART and HeT-A elements in other Drosophila species, we performed in situ hybridization experiments, using both TART and HeT-A probes, on mitotic and polytene chromosomes of D. simulans, D. sechellia, D. mauritiana, D. yakuba and D. teissieri. We found that TART and HeT-A probes hybridize at specific heterochromatic regions of the Y chromosome in all Drosophila species that we analyzed.     

Developmental and tissue-specific accumulation pattern for the Drosophila melanogaster TART ORF1 protein

The TART, HeT-A, and TAHRE families of Drosophila non-LTR retrotransposons specifically retrotranspose to telomeres to maintain telomeric DNA. Recent evidence indicates that an RNA interference mechanism is likely to regulate TART, HeT-A, and TAHRE retrotransposition, but the developmental and tissue-specific expression of telomeric retrotransposon proteins has not previously been investigated. We have generated antisera against TART ORF1 protein (ORF1p) and used these antisera to examine the pattern of TART ORF1p expression in Drosophila melanogaster. We detected TART ORF1p throughout most of development and observed particularly high levels of protein in late larval and pupal stages. In late-stage larvae, ORF1p accumulates in brain and imaginal discs tissues, rather than in terminally differentiated larval tissues. Accumulation of ORF1p in imaginal discs is intriguing, since TART antisense RNA has previously been detected in imaginal discs, and we discuss the implications of these findings for TART regulation.     

Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres.

In most eukaryotes telomeres are extended by telomerase. Drosophila melanogaster, however, lacks telomerase, and telomere-specific non-LTR retrotransposons, HeT-A and TART, transpose specifically to chromosome ends. A Drosophila strain, Gaiano, that has long telomeres has been identified. We extracted the major Gaiano chromosomes into an Oregon-R genetic background and examined the resulting stocks after 60 generations. In situ hybridization using HeT-A and TART sequences showed that, in stocks carrying either the X or the second chromosome from Gaiano, only the Gaiano-derived chromosomes display long telomeres. However, in stocks carrying the Gaiano third chromosome, all telomeres are substantially elongated, indicating that the Gaiano chromosome 3 carries a factor that increases HeT-A and TART addition to the telomeres. We show that this factor, termed Telomere elongation (Tel), is dominant and localizes as a single unit to 69 on the genetic map. The long telomeres tend to associate with each other in both polytene and mitotic cells. These associations depend on telomere length rather than the presence of Tel. Associations between metaphase chromosomes are resolved during anaphase, suggesting that they are mediated by either proteinaceous links or DNA hydrogen bonding, rather than covalent DNA-DNA bonds.     

Drosophila telomeres: A variation on the telomerase theme

In Drosophila, the role of telomerase is carried out by three specialized retrotransposable elements, HeT-A, TART and TAHRE. Telomeres contain long tandem head-to-tail arrays of these elements. Within each array, the three elements occur in random, but polarized, order. Some are truncated at the 5' end, giving the telomere an enriched content of the large 3' untranslated regions, which distinguish these telomeric elements from other retrotransposons. Thus, Drosophila telomeres resemble other telomeres because they are long arrays of repeated sequences, albeit more irregular arrays than those produced by telomerase. The telomeric retrotransposons are reverse-transcribed directly onto the end of the chromosome, extending the end by successive transpositions. Their transposition uses exactly the same method by which telomerase extends chromosome ends--copying an RNA template. In addition to these similarities in structure and maintenance, Drosophila telomeres have strong functional similarities to other telomeres and, as variants, provide an important model for understanding general principles of telomere function and evolution.