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Home , Long terminal repeat, Retrotransposon, Subtelomere

AKE PCA ETR:PERSPECTIVE FEATURE: SPECIAL SACKLER

Retrotransposons that maintain ends

Mary-Lou Pardue1 and P. G. DeBaryshe Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Edited by Joan Curcio, Wadsworth Center, New York State Department of Health, Albany, NY, and accepted by the Editorial Board June 28, 2011 (received for review January 30, 2011)

Reverse transcriptases have shaped in many ways. A remarkable example of this shaping is found on of the genus Drosophila, where have a vital role in chromosome structure. Drosophila lacks ; instead, three - specific retrotransposons maintain chromosome ends. Repeated transpositions to chromosome ends produce long head to tail arrays of these elements. In both form and function, these arrays are analogous to the arrays of repeats added by telomerase to in other . Distantly related Drosophila exhibit this variant mechanism of telomere maintenance, which was established before the separation of extant Drosophila species. Nevertheless, the telomere-specific elements still have the hallmarks that characterize non- (non-LTR) retrotransposons; they have also acquired characteristics associated with their roles at telomeres. These telomeric retrotransposons have shaped the Drosophila , but they have also been shaped by the genome. Here, we discuss ways in which these three telomere-specific retrotransposons have been modified for their roles in Drosophila chromosomes.

| chromosome evolution | | | Y chromosome

ells invest an unexpected amount HeT-A, TART, and TAHRE: A Ménage cessive transpositions produce long arrays of their resources in what might à Trois? of these elements, all oriented with their Cseem to be relatively insignificant Like other complex repeat sequences, 5′ ends toward the end of the chromo- parts of the chromosome, their telomere database entries are prone to some, and many showing some truncation telomeres. Multicellular tend misassembly and require special verifica- of their 5′ end (Fig. 2 is an example). HeT- to have 10 or more kb of telomere repeats tion. In fact, D. melanogaster is the only A, TART, and TAHRE probably have on each chromosome end; even unicellular member of the genus whose genome has equivalent roles in telomere arrays, be- eukaryotes have a few hundred base pairs been sequenced thoroughly enough to al- cause all three retrotranposons seem to be of telomere repeats per end. Telomere low reliable conclusions about the pop- distributed randomly in telomere arrays. length is regulated in species- and ulation distribution of telomere elements. type-specific ways; it is dynamic and can be Therefore, this section will be limited to The Three Telomere-Specific Elements Share influenced by diverse factors, including this species, with brief comments on other Characteristics Not Seen in Other Jockey Clade environment, genetic background, stress, species at the end. Non-LTR Retrotransposons. These charac- and health (1–3). Telomere maintenance HeT-A, TART, and TAHRE are the only teristics include: (i) Telomere elements is complex. It is also essential for cell telomere-specific retrotransposons found transpose only onto chromosome ends. replication and genome integrity. in D. melanogaster (Fig. 1). All are non- There is no apparent DNA sequence In this Perspective, we discuss a dra- LTR retrotransposons belonging to the specificity for the attachment site; they matic alternative to the almost universal jockey clade (Fig. S1), an abundant group transpose onto the 5′ ends of other ele- use of telomerase to maintain telomeres. of non-LTR retrotransposons scattered ments on the chromosome end whether Drosophila lacks telomerase. Instead, spe- over both euchromatic and heterochro- those ends are intact or broken, including cialized non-LTR retrotransposons (6–13 matic regions of the Drosophila genome. broken chromosome ends that have lost all kb long) extend telomeres by transposing These three retrotransposons are the telomere and sometimes, subtelomere se- onto chromosome ends to form head to only members of this clade found in telo- quences (7–10). (ii) Other transposons are tail repeats (4, 5). Despite the apparent meres. Potentially active copies are found not found in telomere arrays (11). (iii) The differences, Drosophila telomeres, like nowhere except telomeres, although de- telomere-specific elements are not found other telomeres, are extended by reverse cayed fragments are present in other in euchromatic DNA unless that DNA is of an RNA template— heterochromatin. a newly transcribed copy of a telomeric It Is Significant That HeT-A, TART, and TAHRE —and share many other This paper results from the Arthur M. Sackler Colloquium of Are Non-LTR Retrotransposons: The Mech- “ characteristics of telomerase telomeres. the National Academy of Sciences, Telomerase and Retro- anism by Which This Set of Retroelements transposons: Reverse Transcriptases That Shaped Ge- For example, Drosophila telomeres are Transposes onto Chromosomes Is Basically nomes” held September 29–30, 2010, at the Arnold and comparable in length with those of other Equivalent to That Used by Telomerase. Mabel Beckman Center of the National Academies of Sci- metazoans and are much longer than ences and Engineering in Irvine, CA. The complete program Non-LTR elements enter the nucleus as and audio files of most presentations are available on telomeres of most single-celled eukar- RNA, the 3′ end of this RNA associates the NAS Web site at www.nasonline.org/telomerase_and_ yotes. Accordingly, the Drosophila telo- with a nick in the chromosome, and its retrotransposons. fi mere-speci c retrotransposons provide an reverse transcription is primed off the 3′ Author contributions: M.-L.P. and P.G.D. designed re- unexpected link between chromosome OH of the nicked DNA, linking the new search, performed research, analyzed data, and wrote the paper. structure and transposable elements, a DNA to the chromosome (6–8). Although fl link that raises questions about the evo- we lack iron-clad proof, there is plenty of The authors declare no con ict of interest. lution of both and specifically about evidence that telomeric retrotransposon This article is a PNAS Direct Submission. J.C. is a guest ed- mechanisms underlying telomere length itor invited by the Editorial Board. RNA associates with the end of the DNA 1 homeostasis in Drosophila in which the To whom correspondence should be addressed. E-mail: rather than with an internal nick like other [email protected]. retrotransposon repeats are more than elements. Thus, each transposition of This article contains supporting information online at three orders of magnitude longer than a telomeric element adds a new end to the www.pnas.org/lookup/suppl/doi:10.1073/pnas.1100278108/-/ telomerase repeats. DNA, extending the chromosome. Suc- DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100278108 PNAS | December 20, 2011 | vol. 108 | no. 51 | 20317–20324 Downloaded by guest on September 29, 2021 tionary conservation of the three elements suggests that each one makes a contribu- tion to maintaining Drosophila telomeres. As mentioned earlier, the apparently random distribution of HeT-A, TART, and TAHRE in telomere arrays argues that they are essentially equivalent in their ability to form telomere . How- ever, there is strong evidence that the three elements collaborate in different ways in transposing to chromosome ends. In diploid somatic cells, the HeT-A Gag specifically localizes to chromo- some ends in interphase nuclei. TART Gag moves into nuclei in these cells but moves to chromosome ends only if assisted by HeT-A Gag (16). TAHRE Gag is pre- dominantly cytoplasmic but localizes ad- jacent to the nuclear membrane; it also requires HeT-A Gag for localization to Fig. 1. Telomeric retrotransposons from D. melanogaster and D. virilis (approximately to scale). Ma- telomeres (17). The colocalization of genta, 5′ and 3′ UTRs of HeT-A and TAHRE; blue, 5′ and 3′ UTRs of TART; white, Gag and Pol ORFs; gold TART and TAHRE Gags with HeT-A Gag ′ ′ mel ′ arrows in 5 and 3 UTRs of TART elements (see Figs. 3B and 4 and accompanying text), PNTRs; (A)n,3 provides an opportunity for HeT-A to use oligoA; bent arrows, transcription start sites for full-length sense-strand RNA (note that, for HeT-Amel the RT of either of the other two ele- vir and TART , the element transcribed is immediately downstream of the element shown); asterisks above ments. It is possible that the choice of RT TART elements, start site for short sense-strand RNA; asterisks below TARTs, start site for nearly full- may depend on cell type: in oocytes, length antisense RNA (not determined for TART-C). The length of 5′ UTRs in TARTmel elements is ex- tremely variable. Those lengths shown here representing the three subfamilies were the first to be se- TAHRE RNA but not TART RNA is quenced; other members of these subfamilies have shorter or longer 5′ UTRs. 5′ PNTR extends to the 5′ coexpressed with HeT-A (15). end of the element; thus, the length of any element’s3′ PNTR is defined by the length of its 5′ PNTR. Although the TARTvir 3′ UTR is much shorter than the 3′ UTR of the other elements, it is more than two Drosophila Cell Interactions with Telomere times the length of the 3′ UTR of nontelomeric jockey clade elements that we have analyzed. TARTvir Pol Elements Differ from Their Interactions with ORF also has a 3′ extension of ∼1.2 kb, with no obvious motifs to indicate its function (14). This sequence is Nontelomeric Retrotransposons. Although not seen in the other elements and might do double duty as 3′ UTR when the element forms telomere Gags of telomeric elements are efficiently mel – vir DNA. Elements shown are HeT-A , U06920, nucleotides 1,015 7,097; HeT-A , AY369259, nucleotides localized to chromosome ends, other jockey – mel mel mel vir 7,211 13,612; TART -A, AY561850; TART -B, U14101; TART -C, AY600955; TART , AY219709, clade Gags are almost entirely retained in nucleotides 4,665–13,208; and TAHRE, AJ542581. the cytoplasm, even when HeT-A Gag is present (18). We suggest that cytoplasmic the end of a broken chromosome. Frag- and its RT is similar to that of TART (12). retention of the Gags of nontelomeric ret- ments of the elements (mostly from the Homologs of HeT-A and TART have been rotransposons is one layer of cellular de- UTRs) have been found in nontelomere identified in D. virilis (>40 Myr separa- fense against parasitic elements, whereas heterochromatic regions, but these frag- tion from D. melanogaster), showing that telomeric Gags and their host cells have ments seem to have been passively moved these elements probably have been main- coevolved a nuclear localization mecha- to these locations rather than actively taining telomeres since before the sepa- nism to facilitate telomere maintenance. transposed (discussion of Y ration of the extant Drosophila species (13, The Rasi RNAi mechanism that protects below) (11). (iv) Telomere elements have 14). TAHRE elements have been detected cells from parasitic DNA is also involved in atypical, very long UTRs. in several species of the melanogaster modulating the rate of transposition of species subgroup (separation = 10–15 telomeric elements in oocytes (19, 20), Although They Apparently Share a Specific Myr) and may well be present in more suggesting that this cellular defense has Role in Telomere Maintenance, HeT-A, TART, distantly related species (15). This evolu- been modified for telomere regulation. and TAHRE Are Surprisingly Different from Each Other. For example, most, if not all, nontelomeric jockey clade elements en- code a (RT). Both TART and TAHRE encode this enzyme, but HeT-A does not have an RT in any species studied. This lack of self- sufficiency might be thought to diminish the efficiency of HeT-A transposition, but in all the D. melanogaster stocks that we Fig. 2. The four most proximal elements in the sequenced array from the XL telomere drawn ap- have studied, both HeT-A and TART are proximately to scale. All elements are HeT-A, and each element is joined by its 3′ oligoA to its proximal present, and HeT-A is always much more neighbor. The most proximal element, HeT-A {}4,800, is complete, two elements are truncated in the 3′ abundant than TART, no matter how UTR, and one element is truncated in the ORF. Elements are identified by FlyBase identifier number. ′ ′ ′ much telomere DNA the stock contains Dark blue, 3 UTR; light blue, ORF; magenta, 5 UTR; white, string of Tags on the 5 end of complete element {}4,800; gold, beginning of the subtelomere region; bent arrows, transcription start sites (each (11). TAHRE is rare, although it seems to arrow indicates a cluster of three closely spaced sites at the 3' end of the element). The start site on {} combine the best features of the other two 5,504 initiates transcription of {}4,800, a transposition-competent element. Other starts will not produce elements because its UTR and gag se- productive transcripts. Physical mapping of BACs from this stock indicates that this telomere extends quences are very similar to those of HeT-A >100 kb further to the left (51), but no sequence is available.

20318 | www.pnas.org/cgi/doi/10.1073/pnas.1100278108 Pardue and DeBaryshe Downloaded by guest on September 29, 2021 For Multicopy Elements, Phylogenetic Studies However, retrotransposon telomeres have at sites within the 3′ end of the upstream Provide Important Information About Evo- an added responsibility; they must preserve element (26, 27). Thus, each new full- lutionary Origins and the Functional Impor- at least some intact retrotransposons to length sense-strand RNA has a very short tance of Conserved Features. We began supply new transpositions to maintain copy of 3′ sequence, including an oligoA phylogenetic studies of Drosophila telo- chromosome ends, because we have found tail, added to its 5′ end as a Tag (23). meres by selecting λ-phage clones of DNA no other source of complete telomere Although three of the four elements in from D. yakuba (5–15 Myr separation elements. Analysis of sequence from Fig. 2 are 5′ truncated, all have intact 3′ from D. melanogaster). Importantly, telomere arrays reveals a statistically sig- ends carrying transcription start sites (in- λ-phage clones carry enough contiguous nificant overabundance of intact elements, dicated by bent arrows). Each appears DNA to contain entire elements and ele- evidence for protection of transposition- capable of driving transcription of its ment junctions, eliminating the possibility competent elements (11, 23). downstream neighbor, even if the neigh- of misassembly of smaller sequences. Low D. melanogaster and D. virilis HeT-A and bor is truncated. However, our RNA stringency hybridization with the most TART have two unusual mechanisms to studies show that most, if not all, detec- mel conserved part of TART RT identified protect essential 5′ sequences from termi- tible RNA is transcribed from full-length clones of D. yakuba TART (21), which also nal erosion. D. melanogaster HeT-A (HeT- elements, suggesting some regulation of contained HeT-A in head to tail arrays. A Amel) and D. virilis TART (TARTvir) add either transcription or turnover (28). Fur- similar strategy identified TART and HeT- pilfered redundant sequences to the 5′ end thermore, statistical overabundance of A from D. virilis (40–60 Myr separation of the transposing RNA to buffer sequence complete elements in telomere DNA (11) from D. melanogaster) (13, 14). Using the loss of the transposed element (Table 1 and and complete lack of Tags on partial ele- cloned sequences to characterize these Fig. 3A). D. melanogaster TART (TARTmel) ments support the idea that only complete elements in intact flies and cultured cells, also adds expendable sequence to its 5′ end elements are capable of transposing. In we found strong conservation of many (Table 1 and Fig. 3B); sequence copied Fig. 2, only HeT-A {}4,800 is complete and unusual features across the genus, e.g., the from the element’s own 3′ UTR during expected to transpose. discussion of their 5′ end protection be- reverse transcription (24, 25). A Tag on the RNA can be reverse- low. We conclude that retrotransposons D. virilis HeT-A (HeT-Avir) is an enigma transcribed onto the chromosome, be- constitute a robust mechanism of telo- (24). The HeT-Avir promoter, like the coming an extension of the 5′ UTR of the mere maintenance that may have arisen promoter of most non-LTR retrotrans- new element (Fig. 3A). This Tag provides well before the separation of the posons, is located in the 5′ UTR. Tran- an expendable sequence to buffer loss of Drosophila genus. scription starts upstream of the promoter, essential 5′ sequence from chromosome- Do other Drosophila species have telo- and the 5′-most sequence of the RNA is end erosion. If a new transposition caps the meric elements not in the HeT-A, TART, essential for transcription of the new ele- chromosome end before the Tag has vir and TAHRE families? One element (U ment after transposition. There is no eroded completely, the truncated Tag re- in our D. virilis telomere clones) has HeT- mechanism for adding buffering sequence; mains as a 5′ extension of the element. A A 5′ and 3′ UTR sequences but lacks nevertheless, D. virilis telomere arrays new Tag is added each time an element is a Gag gene; instead, it has a complete and contain a significant fraction of complete transcribed, and this Tag will be attached open RT gene (13). Neither molecular HeT-As. Thus, HeT-Avir must have an- to the existing string of truncated Tags. studies of our D. virilis stock nor analysis other mechanism to protect its 5′ end. Thus, an element can have a 5′ string of of Genome Project scaffolds reveals the variably truncated tags, providing evidence vir On an evolutionary timescale, the dif- multiple U elements expected of a bona ferent mechanisms in this small sample that it has transposed several times. For fide replication-competent element. Also, show an unexpected variation of end pro- example, HeT-A {}4,800 (Fig. 2) has nine analysis of sequence from other Drosoph- tection. However, only transposition- Tags of variable lengths on its end, showing fi ila species has identi ed a number of pu- competent elements can give rise to line- that it has transposed at least nine times. tative telomere retrotransposons (22); ages of new elements, which provides a very When this element is next transcribed, the functional studies of these sequences will strong drive for evolving efficient 5′-end RNA will have a Tag of terminal sequence be necessary to determine if they are bona protection. The diversity of mechanisms from HeT-A {}5,504 added to the existing fi de telomeric elements and how they re- seen in these studies seems to be a result Tags (in white). The presence of Tags fi ′ late to HeT-A, TART, and TAHRE. of this drive. con rms that the 5 end of an element is functionally complete. Although some fi Telomere-Speci c Retrotransposons HeT-Amel and TARTvir (and Maybe TAHRE) replicatively complete elements might lack Have Evolved Innovative Ways to Share an Unusual Promoter Architecture That Tags, we have not seen an element without Protect Essential Sequences at Their ′ Tags that has the complete 5′ UTR seen on ′ Adds Buffering Sequence to the 5 End of the 5 Ends RNA Transcript. Promoter sequences slightly elements with Tags. mel Because telomere elements are reverse- upstream of the 3′ end of each of these two The promoter used by HeT-A and vir transcribed onto the chromosome end, elements drive transcription, not of that TART has not been found in other non- each newly transposed element is oriented element, but of its downstream neighbor LTR elements but resembles the pro- so that its 5′ end is also the end of the (Figs. 2 and 3A). Transcription starts moters of LTR retrotransposons and ret- chromosome. Thus, the element is subject roviruses. Promoters and transcription to terminal erosion until another element start sites of LTR elements are in the 5′ transposes to take over the terminal posi- LTR (29). For two adjacent HeT-Amel (or Table 1. Mechanisms to add extra 5’ tion. Established Drosophila telomeres TARTvir) elements in a telomere array, the sequence consist of many kilobases of HeT-A, TART, 3′ end of the upstream element is essen- and TAHRE; many of these elements are Element Mechanism tially identical to the 3′ end of the down- variably 5′ truncated, showing that these stream element; furthermore, the 3′ end of HeT-Amel Start transcription in chromosome ends, like ends maintained by the upstream neighbor not only looks like and TARTvir upstream element telomerase, are dynamic (11). We believe but temporarily acts like a 5′ LTR for the HeT-Avir Unknown that the incomplete elements in Drosophila downstream neighbor, because it contains TARTmel 2nd reverse transcription telomeres also fulfill the roles carried out the transcription start site. This pseudo- of 3’ PNTR by telomerase repeats in other organisms. LTR promoter differs from bona fide LTR

Pardue and DeBaryshe PNAS | December 20, 2011 | vol. 108 | no. 51 | 20319 Downloaded by guest on September 29, 2021 that, like LTRs, the two TARTmel repeats coevolve because both are reverse-tran- scribed from only one of two repeats in the RNA. However, in TARTmel (a non-LTR element), the coevolution mechanism dif- fers in its details from those in LTR ele- ments; most notably, the 3′ TARTmel repeat does not extend to the 3′ end of the element. Thus, the TARTmel repeats are not strictly terminal, and we refer to them as perfect nonterminal repeats (PNTRs). In TARTmel, the transcription start site for the full-length sense strand lies in the 5′ PNTR, just upstream of ORF 1. Despite the marked sequence differences in the ′ Fig. 3. Mechanisms for adding buffering 5 sequence. (A) Sequence copied from upstream neighbor. subfamily UTRs, all three subfamilies Used by HeT-Amel and TARTvir. Telomere segment with a complete HeT-Amel flanked by other HeT-As. share the same site. Comparing sub- Transcription starts at the bent arrow in the upstream element and continues through the complete fi element. The resulting RNA (black line) has a Tag of the last nucleotides of the upstream element. On families by BLAST (blastn), we nd small transposition, this Tag will become the 5′ end of the new element, undergo erosion, and if transposed islands of nucleotide similarity surround- again, be internalized into the string of variably eroded Tags indicated by the gray box at the 5′ end of ing the sense-strand start sites, suggesting the complete element. (B) Sequence copied from the 3′ UTR of transposing RNA. Used by TARTmel. that these sequences have been con- mel fl ′ Telomere segment with a complete TART anked by distal TART and proximal HeT-A.(A)n,3 oligoA served, while surrounding UTR sequences in DNA; AAAAAA, polyA tail on RNA; gold arrows, PNTRs; other annotation as in Fig. 1. Transcription have diverged. starts at the bent arrow and produces RNA with a very short 5′ UTR. When this is reverse-transcribed onto These sequence analyses suggested that ′ ′ the chromosome end, the RT jumps back to the 3 UTR and copies sequence to extend the 5 UTR (Fig. 4). all TARTmel RNA transcripts have a very short 5′ UTR, which is extended during promoters in one important way: the Tag TARTmel RNA. Maxwell et al. (27) used transposition by repeating the reverse ′ transcription of the 3′ PNTR (Figs. 3B and added to the new transcript does not RACE analysis to determine the 5 end of fi contain the complete promoter sequence. TARTmel , and identify transcription 4). Speci cally, we postulated that when the reverse transcription of the RNA onto the Therefore, the newly transposed element start sites, whereas we identified promoter chromosome reaches the 5′ end of the does not possess its own promoter and activity with reporter constructs (24–26). RNA, the RT makes a template jump to the remains a non-LTR element. Clearly, Both methods gave the same result, iden- identical sequence in the 3′ end of the 3′ HeT-Amel and TARTvir pay a price for this tifying only one site for full-length sense PNTR (Fig. 4). It then continues to extend arrangement; their transposition is possi- RNA, a site conserved in all three sub- the 5′ UTR of the new element by making families of TARTmel (Fig. 1). These results ble only if two sister elements occur in a second copy of the 3′ UTR, thus elon- were supported by the presence at that site tandem on the telomere. Strong selection gating the 5′ UTR of the transposed ele- for maintaining transposition-competent of a very good match to the downstream ′ ment and incorporating any changes that elements must balance the cost of this promoter element and initiator of the 5 have occurred in the 3′ PNTR (25, 31). The unusual arrangement. UTR promoters typical of many non-LTR fi proposed mechanism differs from that used TAHRE has not been identi ed in D. retrotransposons. This result is paradoxi- by LTR elements to maintain the identity of virilis, and there is not enough sequence cal: the transcription start is ∼75 nt 5′ of the fi mel mel their two ends. Speci cally, the TART information on TAHRE to characterize start codon of ORF 1 (Fig. 3B), but only RT must be capable of making a template mel ′ a possible buffer mechanism. However, the one reported TART 5 UTR, 33 nt long, jump from the 5′ end of the RNA back to strong similarity to HeT-A UTR sequences, is short enough to have been transcribed ′ mel the end of the 3 PNTR. It must also dis- supported by evidence that TAHRE has from this start site. (The few available sociate the 3′ PNTR cDNA from its RNA mel mel ′ a3′ promoter similar to that of HeT-A , TART 5 UTRs range from 33 to 3,934 template to make a second copy of the mel ′ suggests that TAHRE shares the HeT- nt.) Whence came all other 5 UTRs? RNA. There is reason to expect the mel A buffering mechanism (15). We have proposed an explanation using TARTmel enzyme can accomplish this dis- a model based on some of the other un- sociation because unlike the better known TARTmel also Adds a Protective 5′ Sequence usual features of TARTmel UTRs (27). mel retroviral enzymes, RTs from at least two but Does so by Making a Second Copy from There are three TART subfamilies, A, non-LTR elements (R2Bm and mouse L1) ′ Its Own 3 UTR When It Is Reverse-Tran- B, and C (Fig. 1). As with other telomeric do make template jumps (31–33). The scribed onto the Chromosome. D. mela- retrotransposons, coding sequences in R2Bm enzyme is also able to separate nogaster TART apparently has evolved these subfamilies are somewhat variable. the cDNA from its RNA template ′ a mechanism for maintaining its 5 end that In contrast to HeT-A subfamily UTRs, without destroying the template, whereas differs, in all details but not in principle, which anomalously are not much more retroviral RNaseH degrades RNA bound from the one found for D. virilis TART. variable than their coding regions (11), to cDNA (34). mel Surprisingly, TART does not have TARTmel UTRs differ markedly between The second time around, the continu- the pseudo-LTR promoter used by both subfamilies. Nevertheless, the UTRs of all ing reverse transcription of the TARTmel its D. virilis homolog and its HeT-A partner TARTmel subfamilies share one unusual 3′ PNTR adds a sacrificial 5′ sequence in D. melanogaster. (This mechanism characteristic: each contains a pair of long that, like the Tags of TARTvir and HeT- might be unfavorable, because TARTmel is repeat sequences (30), one in the 5 ′UTR Amel, can be lost to terminal erosion greatly outnumbered by HeT-Amel and is and its match in the 3′ UTR (Fig. 1). without affecting the transposition less likely to have another TART as an Within individual elements, these competence of the transposed element. upstream neighbor.) Extensive searches repeats are clearly evolving together (25), The extreme variability in the length of have found only a single start site for like the LTRs of and LTR the 5′ UTR in genomic TARTmel ele- transcription of full-length sense-strand retrotransposons (29). We have proposed ments could be explained by variable

20320 | www.pnas.org/cgi/doi/10.1073/pnas.1100278108 Pardue and DeBaryshe Downloaded by guest on September 29, 2021 the sense-strand promoter in the two spe- mosome has a small transition zone at the cies, suggests that the antisense RNA is proximal edge of the array where there are important to the biology of the element. some fragments of nontelomeric elements However, no role is known for the tran- mixed with fragments of telomeric ele- script. TARTmel is regulated by RasiRNA in ments. We do not include these transition the female germ line, but there is no evi- zones in our discussion of telomere arrays. dence that the large antisense transcript is The most distal element in each array has involved (19). been truncated by cloning and is also omitted. There is no available information The Sequences of Telomeric on the organization of the most extreme Retrotransposon Arrays Provide end of Drosophila telomeres. a Chronological Record of Dynamic As explained below, the existing data Activity at Chromosome Ends justify positing three mechanisms for the Telomere arrays are elongated by succes- maintenance of telomere-length homeo- sive transpositions onto chromosome ends. stasis: small-scale end erosion averaging ∼ ′ Thus, each element is older than the ele- 20 nt between transpositions, large-scale Fig. 4. Proposed mechanism for extending the 5 ments distal to it. The sequence information end of D. melanogaster TART. Transcription starts terminal deletions that can encompass is rich in detail, because each element has part or all of the telomere, and balancing (bent arrow) near the ATG of ORF 1 (gag), pro- fi ducing a transposition intermediate RNA (dashed several identi ers: (i) number of A residues sporadic transpositions that add large black line) lacking most of the 5′ UTR. This RNA has copied when reverse transcription of that segments of DNA and renew the supply of a small piece of the parent element’s5′ PNTR (short element was initiated, (ii) subfamily se- transposition-competent elements. gold arrow) and a complete 3′ PNTR (long gold quence of the element, (iii) extent of 5′ arrow). Steps 1–3 show the RNA as it is reverse- truncation, and (iv) amount of nonessential HeT-A Elements in the Assembled Arrays transcribed into DNA on the chromosome end. sequence remaining on the 5′ end of com- Provide SufficientDatatoMakeQuantitative (Step 1) The polyA tail associates with the chro- plete elements. In our analyses, these Assessments of Telomere Dynamics in D. mosomal DNA (magenta), and RT begins to copy identifiers have allowed unique identifica- melanogaster. The ∼100-kb sequence ana- the RNA. The gray oval represents pro- tion of elements. Furthermore, although lyzed contains 4, possibly 5, intact and 10 or posed to hold the RNA in a conformation that ′ brings the 5′ PNTR sequence into proximity to the these sequences are highly repetitive, there 11 5 -truncated HeT-A elements from six 3′ end of the 3′ PNTR (omitted for clarity in later is enough microheterogeneity to identify subfamilies as well as three 5′-truncated steps). (Step 2) When RT reaches the 5′ end of the products of recombination (except for ex- and two intact TART elements. There are transcript, it makes a template jump back to the changes within the most precisely aligned no TAHRE elements. This distribution is matching 3′ end of the 3′ PNTR. (Step 3) RT dis- regions). We have seen no evidence for consistent with the relative proportions of sociates the RNA–DNA complex and recopies some significant recombination. Thus, unlike te- the three elements measured in different or all of the 3′ PNTR. As a result, the transposed ′ lomerase telomeres with their myriads of D. melanogaster stocks (11). The length of element will have more 5 UTR sequence than the identical repeats, each telomeric element the arrays on XL and 4R suggests that these RNA did and possibly more sequence and longer fi PNTRs than the element from which it was derived. bears a unique DNA ngerprint that allows proximal elements should have been on the determination of its history in the genome chromosome end long enough for signifi- if its hierarchical position therein can cant sequence decay, because they are no termination of reverse transcription, ter- be determined. longer under selection for function. How- fi minal erosion, terminal deletions, or some Even in D. melanogaster, dif culties in ever, this does not seem to be the case. In- combination thereof. correctly assembling long sequences of tact elements are distributed throughout highly repetitive DNA have largely pre- the array, and all appear to be fully func- TARTmel Produces Two Other RNAs, a Small cluded using whole-genome sequencing for tional. Within the bounds of their natural Sense-Strand RNA and a Nearly Full-Length analysis of the organization and possible variability, truncated elements have lost Antisense RNA, Both of Unknown Function. roles of transposable elements in hetero- only 5′ sequence; remaining coding se- In addition to the transposition inter- chromatic regions like centromeres and quence is still open with no evidence of mediate RNA discussed above, TARTmel telomeres. Fortunately, some sequences decay in the reading frame (23). produces two other abundant RNAs of derived from individual D. melanogaster There is enough HeT-A sequence to al- unknown function. We do not believe that BACs are available. We have analyzed se- low statistically robust quantitative analysis ′ quences of a telomeric BAC from 4R and of the overall dynamics of telomere turn- either RNA is involved in 5 end buffering, fi but we mention them here to avoid con- from directed nishing of a scaffold from over for these elements. There are few the telomere of XL. Both the 4R and the fusion. The small sense strand is produced TART data, and we do not concatenate XL sequences begin within their assembled from the site in the 3′ PNTR that is iden- TART with HeT-A, because the two ele- chromosome and extend into the telomere, ments appear to be regulated differently. tical to the start site in the 5′ PNTR. This 3′ thus showing the precise relationship be- Most relevant to their turnover is the site would produce RNA of a few hundred tween these telomere arrays and the rest of fact that, although we do not have enough nucleotides depending on the TARTmel the genome (11). Neither sequence ex- TART 5′ UTR sequence to estimate subfamily. There is a strong transcription tends to the distal end of the telomere, but the average amount of 5′ sequence termination site at the end of the element, together, they contain nearly 100 kb of added during TART transposition, it is which seems to act on both the large and telomeric arrays (76 kb from 4R and 20 kb clearly much longer than a HeT-A Tag; small RNAs (27). A possible product of the from XL). Importantly, both include the hence, details of TART turnover must be ′ 3 start site has been seen on Northern most proximal and therefore, the oldest different (25). blots, but no function is known (28). elements of the array and thus, present mel The start site for the long TART an- the most complete history available of HeT-A Sequence Provides Two Indicators of tisense RNA is similar in location to that of D. melanogaster telomere maintenance. the Relative Rates of Sequence Loss from vir the antisense promoter for TART , shown The terminal arrays on both 4R and Telomeres. These indicators are (i) the in Fig. 1 (25). Conservation of this pro- XL are composed entirely of head to tail length and number of Tags on the 5′ end of moter, despite the marked differences in telomeric retrotransposons. Each chro- each HeT-A and (ii) the distribution of

Pardue and DeBaryshe PNAS | December 20, 2011 | vol. 108 | no. 51 | 20321 Downloaded by guest on September 29, 2021 complete and 5′ truncated elements in the Furthermore, the very shortest Tags are have enough 3′ UTR to provide promoter telomere array. These observations, which overrepresented (18% have the sequence activity for a downstream neighbor, al- describe the results of processes governing TAAA), suggesting that the rate of se- though the shortest element would provide telomere maintenance and renewal, con- quence loss is reduced as Tags are eroded to only weak activity. strain models based on detailed numerical their oligoA tail. There is also one very long Lengths of the truncated elements scale analysis. In turn, statistical analyses help in Tag (68 nt) that is a distinct outlier; the next from 5,892 to 241 bp and have no obvi- distinguishing and judging competing longest is 38 nt. The paucity of long Tags ous correlation with position in the array. conclusions. may be evidence that the two distant tran- The relation of sequence loss to length It is important to note that the sequence scription starts, −93 and −62, are very rarely for these elements is very different from data analyzed here can be used only to used. Alternatively, these longest Tags that seen by analyzing Tag strings, sug- determine relative rates for the different may be subject to more severe erosion, but gesting that these truncations are the processes that we study. This finding is in Tags originating near −31 nt seem to be result of a different process. We suggest contrast to measurements on the rate of strongly protected until they loose several that at least some of this truncation re- loss from broken chromosome ends lacking nucleotides (25). For shorter Tags, the sults from terminal deletions that may telomere sequences. From broken chro- median nucleotide loss is 25 nt, and the occur anywhere within the array, leading mosome studies, several groups (35–37) mean is 23.7 nt (SD = 6.3 nt). to occasional rebuilding of all or part of have determined that, unless healed by a Analysis of the strings of Tags on in- the array (23). telomere retrotransposon, the broken dividual elements yields information on the There is no a priori reason to expect that chromosome end recedes at about ∼70 nt relative rate of transposition onto those some terminal deletions will not remove per fly generation (i.e., between the mea- elements. The narrow limit on Tags per the entire telomere array and possibly, surement of its length in a male and its string (5–9) and Tag string length (69–161 extend farther into the chromosome; fur- length in his son). It is interesting to think nt) indicates that erosion is under some thermore, there is evidence of such dele- about the sequence losses seen in our sort of control; we find neither intact HeT- tions from studies of subtelomeric regions telomere arrays in terms of the times As without Tags nor Tag strings that have in natural populations. Subtelomeric measured for broken ends, but as dis- grown without limit, as they would have if regions have high levels of gene presence/ cussed below, we conclude that sequence not effectively pruned (23). absence polymorphism not seen in the loss from telomere arrays is very different These analyses show that the erosion adjacent euchromatin. At least some of this from the more or less regular, continuous process at the telomere end is more com- structural polymorphism is due to terminal erosion detected on broken ends. Instead, plex than the relatively regular loss de- deletions that were subsequently healed by we conclude that maintenance of estab- scribed by studies of broken chromosomes, transposition of HeT-A, as shown by early lished telomeres involves at least three which may be the simple result of end studies of lethal giant larvae (2) near the 2L processes acting in concert to maintain replication losses. They also show that tip (38) and a recent, more extensive study relatively stable conditions: relatively many, perhaps all, new transpositions of the tip of 3L (39). small-scale terminal erosion, large-scale occur before the terminal Tag has been The loss of long segments of telomeres terminal , and irregularly spaced completely eroded. has been shown to be part of the regulation transpositions (23). The two stochastic processes described of telomere length in other organisms. The here (relatively regular erosion of Tags and first evidence for such regulation came HeT-A Tags Give Evidence for Relatively Small- a tendency to protect the very shortest from studies of terminal rapid deletion in Scale Terminal Erosion. The initial length ones) cannot be the whole story. Given that budding yeast (40). More recently, mam- of a Tag is determined by its transcription one Tag is added per transposition, ter- malian telomeres have been shown to use start site (93, 62, or 31 nt upstream of the minal erosion between transpositions, a similar mechanism (41, 42). Although oligoA of the element providing the pro- measured from individual Tag lengths, is the mechanism for generating terminal moter) plus the oligoA of that element very slow compared with sequence addition deletions may be different in Drosophila, (mean OligoA = 8.3 nt, 95% confidence by new transposition (by a factor of several the result, rapid regulation of telomere interval = 4.6–12.1 nt). Thus, the longest hundred); also, because only complete length is the same. For Drosophila, these initial length would be ∼100 nt. This se- elements seem to be transposition- deletions have a second important con- quence is subject to chromosome-end competent, the existence of multiple tags sequence; deletions remove decayed erosion until another element transposes on complete elements implies that se- elements, allowing replacement by trans- to cap the end of the chromosome. Be- quence addition is two to three orders of position-competent elements when the cause each new transposition adds 6–13 magnitude more rapid than gradual ero- deleted telomere is regenerated by new kb, depending on whether it is HeT-A, sion (23). The result of transposition of transpositions. Deletion and rapid re- TART,orTAHRE, one would expect elements that are much longer than the placement would explain the lack of de- a Tag to be completely lost before the next sequence eroded between transpositions cayed elements found deep in telomere transposition if simple erosion were the should be extensive growth of telomeres, arrays. Replacement by new transpositions only telomere maintenance process. In- but telomere length remains relatively might also select against any nontelomeric stead, arrays have a good proportion of stable within each line studied (11). retrotransposons that had managed to intact elements with truncated Tags on the Analyses of the more truncated elements sneak into the telomere array. 5′ end. Typically, the element has a string in the telomere (below) help explain how of several variably truncated Tags, in- length balance is achieved. HeT-A Arrays That Now Reside in dicating that it has transposed several Centromeric Heterochromatin Have times (23). Analyses of 5′-Truncated Elements Suggest Become Structurally Modified to Be Analysis of Tag sequences allows us to Sporadic Terminal Deletions. In contrast to Very Different from Het-A Arrays in measure the dynamics of erosion on estab- Tag erosion, sequence loss from the 5′-trun- Telomere Regions lished telomeres. On average, the Tags are cated HeT-As is on a much larger scale and Although the telomeric retrotransposons surprisingly short. Their median length, clearly contributes to telomere-length ho- transpose only onto chromosome ends, in including the oligoA tail, is 11 nt, their mean meostasis. Two elements are truncated in situ hybridization identified a large cluster length is 14.0 nt, and the 95% confidence the 5′ UTR, three elements are truncated of HeT-A DNA in the centromere region interval of the mean is 10.7–17.3 nt. in the ORF, and six in the 3′ UTR. All of the D. melanogaster Y chromosome

20322 | www.pnas.org/cgi/doi/10.1073/pnas.1100278108 Pardue and DeBaryshe Downloaded by guest on September 29, 2021 (43). A similar cluster binding antibody to sertion of members of seven families of become a complex array of repeats. Be- centromere-specific was found on nontelomeric transposable elements (45). cause some amplification events apparently the Y chromosomes of other members In contrast, neither amplifications nor in- involved more than a single unit, higher of the melanogaster species subgroup (44). sertion of nontelomeric transposable ele- order repeats arise. Thus, telomeric HeT-A sequences appear ments is seen in telomeric regions, which As a result of both sequence loss and to have been moved into the Y centro- grow entirely by transpositions onto the nucleotide changes, the centromeric HeT- mere before this species subgroup split chromosome ends. (There is one qualifi- A elements have lost much of their protein >13 Mya. The Y chromosome is now cation to this statement; subfamilies of coding capacity. The longest ORFs in metacentric in some of these species and telomeric elements can differ by small in- these elements range from 246 to 558 nt; telocentric in others, but despite this sertions/deletions in both coding and un- in comparison, the shortest complete HeT-A structural reorganization, the HeT-A se- translated regions. Some indels are gag gene is 2,766 nt (23). Whether any quence remains in the centromere region repeats of adjacent sequence; the origin of of these short ORFs in the centromeric of every species. This conserved localiza- others is not obvious. In coding regions, DNA are expressed is an open question. tion suggests that the HeT-A cluster has these indels do not introduce stop codons acquired some role at the centromere, or alter the reading frame and they do not Telomere Elements Nowin the Centromere Clus- possibly forming the kinetochore, affecting affect the A + C strand bias conserved ter Have Been Shaped into Complex Repeats sister chromatid cohesion, or maintaining throughout these elements. In all cases, Similar to Those That Characterize the Hetero- the heterochromatic environment. Men- they are found in multiple elements and chromatic Centromere Regions in Multicellular dez-Lago et al. (45) recently sequenced therefore, do not compromise trans- Organisms. Centromeres in multicellular or- a D. melanogaster BAC that allowed them position of elements.) ganisms are determined epigenetically (47, to characterize the molecular structure of 48); thus, it is not possible to identify cen- this centromeric HeT-A cluster. That Full-Length HeT-A Elements in the Centro- tromeres by sequence alone. Nevertheless, structure reveals dramatic changes from meric Array Have Undergone Extensive Inter- this Y cluster is similar (in size, repeated the structure of telomere arrays. nal Deletion. The centromeric sequences sequence structure, and presence of trans- The Y chromosome BAC contained 159 differ from telomeric sequences not only posable elements) to the only functionally kb of HeT-A DNA. Mendez-Lago et al. in their mechanism of sequence addition characterized centromere in Drosophila,the (45) concluded from their sequence that but also in their mechanism of sequence X chromosome centromere (49), support- this DNA arose from a founder sequence loss. Loss from elements in telomere ar- ing the cytological evidence that this cluster that initially consisted of nine telomere rays is exclusively from their 5′ ends, except is in some way involved in centromeric ac- retrotransposons in a typical telomeric for the small indels noted above. In con- tivity. It is not surprising that the Y chro- head to tail array (Fig. 5). This founder trast, each centromere element has sev- mosome cluster does not share sequences sequence could have been either a Y eral large internal deletions scattered with the centromere of the X chromosome: chromosome telomere moved to the in- through its sequence. There has been little these two chromosomes do not pair nor- terior by an inversion or a segment of rearrangement of the remaining sequence, mally, and a difference in centromere se- telomere from the Y or another chro- most of which is collinear with the ca- quences could well be either a cause or mosome that was inserted into the Y, nonical HeT-A, with relatively few in- aresultofthislackofmeioticpairing. which has a record of accepting sequence versions and rearrangements. Surpris- Y-specific centromere sequences also have from other chromosomes (46). In either ingly, the only regions that are conserved been documented for the mouse Y chro- case, the founder was a typical telomere in every centromere element are the ex- mosome (50). array of about 30 kb. Thus, the sequence treme 5′ and 3′ ends (23). of this BAC provides an unusual oppor- Many deletions in the centromere ele- Conclusion tunity to compare a telomere array that ments are shared with siblings derived from Drosophila telomeres provide a detailed has resided in centromeric heterochro- the same amplification. Thus, these 10 picture of the interactions between a fi matin for signi cant evolutionary time elements and the partial element have metazoan genome and retrotransposons with telomere arrays that have remained on chromosome ends. We find that there are striking differences in the ways that the sequences have been maintained in the two regions.

The Centromeric HeT-A Cluster Has Grown Extensively by Amplifications of Various Parts of the Sequence. The founder se- quence consisted of nine elements. Five of these, four HeT-As and one TART, were extremely 5′-truncated. These elements formed a 3.1 kb repeat that has been am- Fig. 5. Evolution of HeT-A sequences in the centromere region of the Y chromosome, deduced by plified to make up more than 100 kb of Mendez-Lago et al. (45) (not to scale). The bottom diagram shows telomere sequence transposed into relatively homogeneous simple sequence the Y chromosome: eight HeT-A elements (orange arrows) and one partial TART (#4; yellow arrow). repeats typical of the that Elements 1, 2, 3, and 5 are truncated HeT-As, and elements 6–9 are complete HeT-As. The top diagram are abundant in pericentric heterochro- shows 159 kb cloned in the sequenced BAC. The partial elements underwent complex amplifications to matin. The other segment of the founder make up the 18HT satellite, which is partially represented by pentagons and black arrows on the left and fi contained four complete HeT-As. This is not further considered here. The end result of the several ampli cations of the initially complete el- ements is shown on the right (numbering retained from ref. 45 to indicate origin of different parts of the segment has undergone a series of head to fi fi sequence). Elements with two numbers result from ampli cations of parts of two elements. Triangles, tail ampli cations of different regions of nontelomeric retrotransposons (copia, mdg1, diver, F, and 1731) that inserted at various times during the the array to yield 10 elements and another sequential amplifications of this DNA; green boxes, segment of autosomal region 42A transposed into element truncated by cloning. The cen- element 8 and later duplicated. [Based on figure 7 in the work by Mendez-Lago et al. (45) and repro- tromeric cluster has also grown by in- duced with permission from Oxford University Press.]

Pardue and DeBaryshe PNAS | December 20, 2011 | vol. 108 | no. 51 | 20323 Downloaded by guest on September 29, 2021 in coevolving a robust mechanism to that Drosophila telomeres share many, region, produced a repetitive sequence maintain dynamic chromosome ends. if not most, operational characteristics that was shaped, probably passively, into These interactions are not a one-way with telomeres maintained by telomerase the complex DNA repeats that typify street. The retrotransposons have main- in other organisms. This picture of the metazoan centromere regions. tained their identity as retrotransposons, coevolution of telomeric retrotrans- while acquiring other characteristics posons with the genome is strengthened ACKNOWLEDGMENTS. This work was supported important for their roles at telomeres. by the fate of those retrotransposons that, by National Institutes of Health Grant GM50315 The end result of this coevolution is afterbeingmovedintothecentromere (to M.-L.P.).

1. Blackburn EH (2001) Switching and signaling at the 19. Savitsky M, Kwon D, Georgiev P, Kalmykova A, encoded by the R2 retrotransposon. JMolBiol374: telomere. Cell 106:661–673. Gvozdev V (2006) Telomere elongation is under the 322–333. 2. Greider CW (1998) Telomerase activity, cell prolifer- control of the RNAi-based mechanism in the Dro- 35. Biessmann H, Carter SB, Mason JM (1990) Chromosome ation, and . Proc Natl Acad Sci USA 95:90–92. sophila . Dev 20:345–354. ends in Drosophila without telomeric DNA sequences. 3. Epel ES, et al. (2004) Accelerated telomere shortening in re- 20. Shpiz S, Kwon D, Rozovsky Y, Kalmykova A (2009) Proc Natl Acad Sci USA 87:1758–1761. sponse to stress. Proc Natl Acad Sci USA 101:17312–17315. rasiRNA pathway controls antisense expression of 36. Levis RW (1989) Viable deletions of a telomere from 4. Pardue ML, DeBaryshe PG (2003) Retrotransposons pro- Drosophila telomeric retrotransposons in the nucleus. a Drosophila chromosome. Cell 58:791–801. vide an evolutionarily robust non-telomerase mecha- Nucleic Acids Res 37:268–278. 37. Mikhailovsky S, Belenkaya T, Georgiev P (1999) Broken nism to maintain telomeres. Annu Rev Genet 37:485–511. 21. Casacuberta E, Pardue ML (2002) Coevolution of the chromosomal ends can be elongated by conversion in 5. Pardue ML, DeBaryshe PG (2011) Adapting to life at telomeric retrotransposons across Drosophila species. . Chromosoma 108:114–120. the end of the line. How Drosophila telomeric retro- 161:1113–1124. 38. Walter MF, et al. (1995) DNA organization and poly- transposons cope with their job. Mobile Genetic Ele- 22. Villasante A, et al. (2007) Drosophila telomeric retro- morphism of a wild-type Drosophila telomere region. ments 1:128–134. transposons derived from an ancestral element that Chromosoma 104:229–241. 6. Luan DD, Korman MH, Jakubczak JL, Eickbush TH was recruited to replace telomerase. Genome Res 17: 39. Kern AD, Begun DJ (2008) Recurrent deletion and gene (1993) Reverse transcription of R2Bm RNA is primed by 1909–1918. presence/absence polymorphism: Telomere dynamics a nick at the chromosomal target site: A mechanism for 23. DeBaryshe PG, Pardue ML (2011) Differential mainte- dominate evolution at the tip of 3L in Drosophila non-LTR retrotransposition. Cell 72:595–605. nance of DNA sequences in telomeric and centromeric melanogaster and D. simulans. Genetics 179: 7. Biessmann H, et al. (1990) Addition of telomere- heterochromatin. Genetics 187:51–60. 1021–1027. associated HeT DNA sequences “heals” broken chro- 24. Traverse KL, George JA, Debaryshe PG, Pardue ML 40. Lustig AJ (2003) Clues to catastrophic telomere loss in mosome ends in Drosophila. Cell 61:663–673. (2010) Evolution of species-specific promoter-associated from yeast telomere rapid deletion. Nat Rev 8. Biessmann H, et al. (1992) Frequent transpositions of mechanisms for protecting chromosome ends by Dro- Genet 4:916–923. Drosophila melanogaster HeT-A transposable elements sophila Het-A telomeric transposons. Proc Natl Acad Sci 41. Wang RC, Smogorzewska A, de Lange T (2004) Homol- to receding chromosome ends. EMBO J 11:4459–4469. USA 107:5064–5069. ogous recombination generates T-loop-sized deletions 9. Levis RW, Ganesan R, Houtchens K, Tolar LA, Sheen FM 25. George JA, Traverse KL, DeBaryshe PG, Kelley KJ, at human telomeres. Cell 119:355–368. (1993) Transposons in place of telomeric repeats at Pardue ML (2010) Evolution of diverse mechanisms for 42. Pickett HA, Cesare AJ, Johnston RL, Neumann AA, a Drosophila telomere. Cell 75:1083–1093. protecting chromosome ends by Drosophila TART Reddel RR (2009) Control of telomere length by a trim- 10. Kahn T, Savitsky M, Georgiev P (2000) Attachment telomere retrotransposons. Proc Natl Acad Sci USA ming mechanism that involves generation of t-circles. of HeT-A sequences to chromosomal termini in Dro- 107:21052–21057. EMBO J 28:799–809. sophila melanogaster may occur by different mecha- 26. Danilevskaya ON, Arkhipova IR, Traverse KL, Pardue ML 43. Agudo M, et al. (1999) Centromeres from telomeres? nisms. Mol Cell Biol 20:7634–7642. (1997) Promoting in tandem: The promoter for telo- The centromeric region of the Y chromosome of 11. George JA, DeBaryshe PG, Traverse KL, Celniker SE, mere transposon HeT-A and implications for the evolu- Drosophila melanogaster contains a tandem array of Pardue ML (2006) of the Dro- tion of retroviral LTRs. Cell 88:647–655. telomeric HeT-A- and TART-related sequences. Nucleic sophila telomere retrotransposable elements. Genome 27. Maxwell PH, Belote JM, Levis RW (2006) Identification Acids Res 27:3318–3324. Res 16:1231–1240. of multiple transcription initiation, , 44. Berloco M, Fanti L, Sheen F, Levis RW, Pimpinelli S 12. Abad JP, et al. (2004) TAHRE, a novel telomeric ret- and splice sites in the Drosophila melanogaster TART (2005) Heterochromatic distribution of HeT-A- and rotransposon from Drosophila melanogaster, reveals family of telomeric retrotransposons. Nucleic Acids Res TART-like sequences in several Drosophila species. the origin of Drosophila telomeres. Mol Biol Evol 21: 34:5498–5507. Cytogenet Genome Res 110:124–133. 1620–1624. 28. Danilevskaya ON, Traverse KL,HoganNC,DeBaryshe PG, 45. Méndez-Lago M, et al. (2009) Novel sequencing 13. Casacuberta E, Pardue ML (2003) HeT-A elements in Pardue ML (1999) The two Drosophila telomeric trans- strategy for repetitive DNA in a Drosophila BAC clone Drosophila virilis: Retrotransposon telomeres are con- posable elements have very different patterns of tran- reveals that the centromeric region of the Y chromo- served across the Drosophila genus. Proc Natl Acad Sci scription. MolCellBiol19:873–881. some evolved from a telomere. Nucleic Acids Res 37: USA 100:14091–14096. 29. Voytas DF, Boeke JD (2002) Mobile DNA II, eds 2264–2273. 14. Casacuberta E, Pardue ML (2003) Transposon Craig NL, Craigie R, Gellert M, Lambowitz AM 46. Koerich LB, Wang X, Clark AG, Carvalho AB (2008) Low telomeres are widely distributed in the Drosophila (American Society for Microbiology, Washington, DC), conservation of gene content in the Drosophila Y genus: TART elements in the virilis group. Proc Natl pp 631–662. chromosome. Nature 456:949–951. Acad Sci USA 100:3363–3368. 30. Sheen FM, Levis RW (1994) Transposition of the 47. Sullivan BA, Blower MD, Karpen GH (2001) Deter- 15. Shpiz S, et al. (2007) Characterization of Drosophila LINE-like retrotransposon TART to Drosophila chro- mining centromere identity: Cyclical stories and fork- telomeric retroelement TAHRE: Transcription, transpo- mosome termini. Proc Natl Acad Sci USA 91:12510– ing paths. Nat Rev Genet 2:584–596. sitions, and RNAi-based regulation of expression. Mol 12514. 48. Malik HS, Henikoff S (2009) Major evolutionary Biol Evol 24:2535–2545. 31. Bibiłło A, Eickbush TH (2002) The reverse transcriptase transitions in centromere complexity. Cell 138: 16. Rashkova S, Karam SE, Kellum R, Pardue ML (2002) Gag of the R2 non-LTR retrotransposon: Continuous syn- 1067–1082. proteins of the two Drosophila telomeric retrotrans- thesis of cDNA on non-continuous RNA templates. JMol 49. Sun X, Le HD, Wahlstrom JM, Karpen GH (2003) Se- posons are targeted to chromosome ends. J Cell Biol Biol 316:459–473. quence analysis of a functional Drosophila centromere. 159:397–402. 32. Bibillo A, Eickbush TH (2004) End-to-end template Genome Res 13:182–194. 17. Fuller AM, Cook EG, Kelley KJ, Pardue M-L (2010) Gag jumping by the reverse transcriptase encoded by the 50. Pertile MD, Graham AN, Choo KH, Kalitsis P (2009) proteins of Drosophila telomeric retrotransposons: R2 retrotransposon. J Biol Chem 279:14945–14953. Rapid evolution of mouse Y centromere repeat DNA Collaborative targeting to chromosome ends. Genetics 33. Babushok DV, Ostertag EM, Courtney CE, Choi JM, belies recent sequence stability. Genome Res 19:2202– 184:629–636. Kazazian HH, Jr. (2006) L1 integration in a transgenic 2213. 18. Rashkova S, Karam SE, Pardue ML (2002) Element- mouse model. Genome Res 16:240–250. 51. Abad JP, et al. (2004) Genomic analysis of Drosophila specific localization of Drosophila retrotransposon Gag 34. Kurzynska-Kokorniak A, Jamburuthugoda VK, Bibillo A, melanogaster telomeres: Full-length copies of HeT-A proteins occurs in both nucleus and cytoplasm. Proc Eickbush TH (2007) DNA-directed DNA polymerase and and TART elements at telomeres. Mol Biol Evol 21: Natl Acad Sci USA 99:3621–3626. strand displacement activity of the reverse transcriptase 1613–1619.

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