MBE Advance Access originally published online on September 21, 2007
Molecular Biology and Evolution 2007 24(11):2535-2545; doi:10.1093/molbev/msm205
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Research Articles |
Characterization of Drosophila Telomeric Retroelement TAHRE: Transcription, Transpositions, and RNAi-based Regulation of Expression
,1
,
* Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia
Department of Molecular Biology, Moscow State University, Moscow, Russia
Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
Centre for Medical Studies of Oslo University, Moscow, Russia
E-mail: allakalm{at}img.ras.ru.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Telomeres in Drosophila are maintained by transpositions of specialized telomeric retroelements HeT-A and TART rather than by the telomerase activity adding short DNA repeats to chromosome ends in other eukaryotes. A novel element TAHRE was previously found in the telomeric regions of the genome of Drosophila melanogaster stock sequenced by the Genome Project. Comparative genomic analysis confirmed by Southern analysis and in situ hybridization of polytene chromosomes reveals conserved TAHRE elements in the genomes of melanogaster subgroup species. Spontaneous attachment of TAHRE retroelement to the broken end of terminally deleted chromosome allows us to consider TAHRE as the third retrotransposon family involved in telomere maintenance in Drosophila. The abundance of TAHRE transcripts in ovaries is strongly upregulated owing to mutations in the RNA interference genes spn-E, aub, piwi, and vasa locus. spn-E mutations eliminate TAHRE-specific short RNAs in the ovaries. These data suggest that TAHRE is a conservative element involved along with HeT-A and TART in telomere maintenance and a target of the RNAi-based system in the Drosophila germ line. This study reveals similar distribution of TAHRE and HeT-A transcripts, which accumulate in the oocyte, whereas TART transcripts localize in nurse cells. Taking into account a common pattern of HeT-A and TAHRE expression, one may consider TAHRE as a source of reverse transcriptase enzymatic activity for HeT-A transpositions in ovaries.
Key Words: Drosophila • telomere • retrotransposon • TAHRE • RNAi
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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In most eukaryotes, telomeric DNA is maintained by the telomerase activity, which is responsible for the synthesis of short 5–11 nucleotide arrays using an RNA component as a template (Greider and Blackburn 1985
; Bryan and Cech 1999). By contrast, telomeres of Drosophila consist of specialized telomeric non–long terminal repeat (LTR) retrotransposons HeT-A, TART, and TAHRE presented at Drosophila telomeres in mixed tandem head-to-tail arrays (Biessmann, Valgeirsdottir, et al. 1992; Levis et al. 1993; Abad et al. 2004b). Retrotranspositions of HeT-A and TART elements in addition to recombination are the mechanisms of Drosophila telomere elongation (Biessmann, Champion, et al. 1992; Sheen and Levis 1994; Mikhailovsky et al. 1999). Retrotransposons are also found in telomeric regions of such diverse organisms as Bombix mori (Okazaki et al. 1995; Takahashi et al. 1997), Chlorella (Higashiyama et al. 1997), and Giardia lamblia (Arkhipova and Morrison 2001); however, in these species, retroelements are targeted to typical short telomeric repeats. Drosophila telomeric retroelements are the first examples of mobile elements that play a vital role in host telomere maintenance. RNA-templated reverse transcriptase (RT) activity of telomerase is per se equivalent to retrotransposition, strongly suggesting a common origin of telomerase and transposable elements (Eickbush 1997; Nakamura et al. 1997). Indeed, the RT domain of telomerase is similar to that found in different non–LTR retrotransposons (Eickbush 1997; Nakamura et al. 1997). Besides a common role in the elongation of degrading chromosome ends, telomeric repeats provide binding sites for the proteins forming specific telomeric chromatin, which protects chromosome ends from the cell repair system and is involved in the processes of chromosome positioning in the nucleus and telomere condensation in meiosis and mitosis (Cooper 2000). Intriguingly, many of these proteins are common to Drosophila and other organisms that use telomerase (Cenci et al. 2005).
Evolutionary, structural, and functional studies of Drosophila telomeric elements are an important part of investigating the telomere biology. Recently, the mechanism of RNA interference (RNAi) has been shown to be involved in the control of telomere length in Drosophila (Savitsky et al. 2006). An RNAi-based mechanism was found to control expression of endogenous transposable elements and their mobility in different species (Wu-Scharf et al. 2000; Aravin et al. 2001; Sijen and Plasterk 2003; Shi et al. 2004; Svoboda et al. 2004; Kalmykova et al. 2005), protecting against a harmful mutagenic influence of mobile elements on the genome integrity. It is noteworthy that expression and transposition of Drosophila HeT-A and TART telomeric elements are also under the control of the RNAi system. Hence, an RNAi-based mechanism may be considered as a negative regulatory system responsible for proper telomere length maintenance in Drosophila.
TAHRE is a novel telomere-specific retroelement discovered in a D. melanogaster genomic sequence analysis (Abad et al. 2004b). Structural peculiarities of this element imply a common origin for the known Drosophila telomeric retrotransposons.
TART has 2 open reading frames (ORFs), encoding Gag and Pol proteins. HeT-A, the most abundant Drosophila telomeric element, contains a single ORF encoding a Gag-like RNA-binding protein, but lacks RT. Both elements have unusually long 3' and 5' untranslated regions (UTRs). TAHRE shares the presence of 2 ORFs with TART; ORF2, encoding RT and endonuclease domains, is similar to that of TART. The 5' UTR, ORF1, and 3' UTR of TAHRE are similar in the corresponding sequences of HeT-A, which was the cause to designate a newly discovered element TAHRE (telomere-associated and HeT-A–related element) (Abad et al. 2004b). It was proposed that a putative ancestral element evolved to provide telomere maintenance in Drosophila. Similarity of TART and TAHRE ORF1 and ORF2 (encoding Gag and Pol proteins, respectively) indicates that these elements have a common ancestor. HeT-A lacks ORF2 and may have derived from a processed copy of TAHRE.
These elements occupying common telomeric regions seem to fulfill different tasks. Prolonged 3' UTRs of HeT-A elements, which are the main structural components of telomeres, might serve as a platform for protein binding to form specific telomeric chromatin (Danilevskaya, Lowenhaupt, and Pardue 1998). It is proposed that RT necessary for HeT-A transposition might be provided in trans, perhaps by TART (Levis et al. 1993; Rashkova et al. 2002) or by TAHRE (Abad et al. 2004b). Although TART copies are much less abundant in the genome than HeT-A and no TART elements are detected in some telomeres (Levis et al. 1993; Abad et al. 2004a), TART is a conserved component of telomeres in distant Drosophila species (Casacuberta and Pardue 2002, 2003b). TART is a primary target of the RNAi controlling system as one dose of a mutant RNAi gene preferentially causes TART, rather than HeT-A, attachments to broken chromosome ends in ovaries (Savitsky et al. 2006). Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, Champion, et al. 1992; Kahn et al. 2000; Golubovsky et al. 2001); TART attachments were also detected (Sheen and Levis 1994). No TAHRE transpositions to chromosome ends were detected. Only HeT-A and TART homologues were previously identified in the genomes of different Drosophila species (Danilevskaya, Tan, et al. 1998; Casacuberta and Pardue 2002, 2003a, 2003b).
Is TAHRE conserved both in D. melanogaster stocks and Drosophila species? Does it transpose to chromosome ends? Is TAHRE a target of the RNAi machinery? This study tries to answer these questions and presents a functional characteristic of the retrotransposon family TAHRE as an equal participant in Drosophila telomere maintenance along with HeT-A and TART.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Drosophila Strains
Strains bearing spindle-E (spn-E) mutations were ru1 st1 spn-E1 e1 ca1/TM3, Sb1 es spn-E1, and ru1 st1 spn-Ehls3987 e1 ca1/TM3, Sb1 es. aub mutants were aubQC42/CyO and aub HN/CyO. vasa locus mutations were vigEP812 and vasPH165. These mutations affect 2 genes, vig and vasa, located at the same locus (Styhler et al. 1998
; Vagin et al. 2004). We used piwi2 and piwi3 mutations. To detect the TAHRE attachments to the broken chromosome end, we used a terminally truncated chromosome with a break in the yellow locus designated yTD. yTD chromosome is lethal. The homologous y ac X chromosome has a deletion of yellow and achaete loci but not of any vital genes. yTD4/y ac line carrying deficiency terminating in the region 
1.3–1.5 kb upstream yellow transcription start site was used. Appearance of flies with black aristae was monitored in this line for 5 successive generations. Drosophila melanogaster stocks y1;cn1 bw1 sp1 (Bloomington Drosophila Stock Center #2057), Oregon, Gaiano, Su(var)2-504 and ru1 h1 th1 st1 cu1 sr1 ca1/TM3, Sb1 (Bloomington Drosophila Stock Center # 4831) were used in the work. Drosophila simulans, Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, Drosophila erecta, and Drosophila orena stocks are from the Gif-sur-Yvette CNRS Center (France) collection.
Northern and In Situ RNA Analyses
Northern analysis of short RNAs was performed as previously described (Aravin et al. 2001). P32-labeled riboprobe corresponding to a sense strand of TAHRE was synthesized. In situ RNA analysis was carried out according to the earlier described procedure (Kogan et al. 2003) using digoxigenin-labeled strand-specific TAHRE riboprobes. Polymerase chain reaction (PCR)–amplified fragments using primers 5'-TAATACGACTCACTATAGGTCAACCTAAATCAAAACTACC-3' and 5'-GGAGGTCATATATTAAAGGGT-3' or 5'-TCAACCTAAATCAAAACTACC-3' and 5'-TAATACGACTCACTATAGGGGAGGTCATATATTAAAGGGT-3' (GenBank sequence AJ542581
[GenBank]
) including T7 RNA polymerase promoter were used as a template for the synthesis of TAHRE sense and antisense riboprobes, respectively. Hybridization with P32 end–labeled oligonucleotide 5'-ACTCGTCAAAATGGCTGTGATA-3' complementary to mir-13b1 was used as a loading control.
Polymerase Chain Reaction and Reverse Transcriptase–Polymerase Chain Reaction Analyses
The following primers were used—TAHRE-specific primers (corresponding to TAHRE sequence AJ542581
[GenBank]
in GenBank): T3', 5'-CCTAAGTCTACAAAATACTAACTAC-3'; T1, 5'-TCATACCGCCCAATCAGCTTACTC-3'; T2, 5'-CTCGTGTATCTGCTGGCGTTTATG-3'; T3, 5'-CAGACGAATCATAAACGCCAGCAG-3'; T4, 5'-TCGCATCACTTCGTCATGATCAGC-3'; T7, 5'-TACCATAATTCTTAGCCGGTCCAAATATAC-3'; and TL, 5'-AGCAATCCCTTCCCATCAATTTGGTTTCAC-3', and yellow-specific primers (corresponding to X06481
[GenBank]
and X04427
[GenBank]
sequences in GenBank): y13, 5'-AATATTTTGTTTCCGCTAGTTATTG-3'; y12, 5'-ATTGGATTTCGATTGGGCGTCAC-3'; y5, 5'-CAGGAGGCTCGTGCATAGAATGC-3'; y9, 5'-GGTTCAGTGTTCGGGTAATCAGGTG-3'; y17, 5'-AAGACGGCGTCACCAAGGGTATC-3'; 7y, 5'-CTTGCGGCGATGGTCATTAGAGC-3'; and yL, 5'-CTGGCTCCAACTATATCGCTCCTGAAGTTTG-3'.
DNA samples were isolated from the F2 progeny of individual yTD4/y ac females with black aristae according to standard methods (Ashburner 1989). Genomic DNAs were used to amplify the junctions between the newly transposed mobile elements and the yellow DNA. PCR was done using different combinations of T3' primer and yellow primers. TAHRE attachment was confirmed by a long-range PCR performed using TL and yL primers. The product of the long-range PCR was cloned in pTR19R (designated as the clone v7 according to the line number) and sequenced.
Reverse transcriptase–polymerase chain reaction (RT-PCR) was done according to the described procedure (Aravin et al. 2001) using pairs of primers corresponding to TAHRE (T7 and T2) and rp49 (5'-ATGACCATCCGCCCAGCATAC-3' and 5'-CTGCATGAGCAGGACCTCCAG-3') as loading controls. cDNA was synthesized using oligo(dT) primer. Results of RT-PCR analysis were evaluated using the program ImageQuant5.0. Histograms display the quantification of at least 3 experiments.
Semiquantitative PCR of Genomic DNA, Southern Analysis, and In Situ Hybridization with Polytene Chromosomes
Semiquantitative PCR was done to compare the abundance of TAHRE elements in the genomes of y1;cn1 bw1 sp1, Gaiano, and Su(var)2-504 stocks. Amplification was done in the presence of dATP-
P33, PCR products were separated in 5% denaturing polyacrylamide gel and visualized by phosphor imager Storm-840 (Amersham Biosciences, Little Chalfont, UK). Linear range of reaction was determined in preliminary experiments. Three pairs of TAHRE-specific primers (T1–T2, T3–T4, and T7–T2) were used in independent experiments. PCR using primers to the unique rp49 gene represents a loading control. Results were evaluated using the program ImageQuant5.0.
For Southern analysis, DNA samples (10 mkg) were digested with restriction enzymes, fractionated through 0.75% agarose gels, and transferred to Hybond-XL membrane (Amersham Biosciences). Hybridization was performed at 65 °C overnight in 0.5 M NaCl, 0.1 M sodium phosphate (pH 7.5), 25 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate (SDS), 50 mkg/ml denatured salmon sperm DNA, and 5x Denhardt's solution. P32-labeled fragments of TAHRE corresponding to nucleotides 5143–6176 (probe 1) or 6144–7148 (probe 2) of GenBank sequence number AJ542581
[GenBank]
were used (fig. 1A). Probe 3 contained nucleotides 1,339–2,306 of TART-A (GenBank accession number DMU02279). rp49 probe contained a fragment of rp49 cDNA corresponding to nucleotides 364–695 of GenBank number Y13939. Southern hybridization of melanogaster subgroup species genomic DNAs with P32-labeled riboprobe corresponding to probe 1 was performed overnight at 65 °C in the same hybridization solution followed by washes at 55 °C with solution containing 10 mM sodium phosphate and 0.2% SDS.
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Fluorescence in situ hybridization (FISH) with polytene chromosomes was performed as described (Lavrov et al. 2004
). DNA probe 1 (fig. 1A) was labeled using Bionick labeling system (Invitrogen, Carlsbad, CA).
Analysis of Promoter Activity
Constructs were made in the CaSpeR-AUG-ß-gal vector. The sequences to be tested, inserted into the polylinker, drive the lacZ reporter gene expression. TAHRE sequence was derived from the element attached to yellow gene. At 414 bp from TAHRE 3' region was PCR amplified with primers 5'-ATGAATTCTCCAGAGTCCACAACAAGCCC-3' and 5'-ATGGATCCTTTGCTGGTGGAGGTACGGAGAC-3' using clone v7 as a template. HeT-A sequence was derived from HeT-A element attached to yellow gene (z2 line). This element is 98% homologous to HeT-A element 23Zn-3 from the earlier described genomic clone (GenBank number U06920
[GenBank]
). PCR using HeT-A–specific primer 5'-TATGCACAACGTCACTTACCTG-3' and y17 primer was done to amplify the junction between yellow gene and the attached HeT-A element. The PCR product was cloned in pTZ19R (clone z2) and was used as a template to amplify 434 bp of HeT-A promoter region with 5'-ATGAATTCATCCATCGCCCGCAACATG-3' and 5'-ATGGATCCTTTGCTGGTGGAGGTACGGAGAC-3' primers. TAHRE and HeT-A PCR fragments were inserted into the EcoRI and BamHI polylinker sites of CaSpeR-AUG-ß-gal. Constructs were verified by sequencing. Drosophila cell culture Schneider 2 was used for transfection performed according to the earlier described procedure (Kalmykova et al. 2004). ß-Galactosidase activity in the cells transfected by CaSpeR-AUG-ß-gal and in "no-DNA" control cells was measured in each series of experiments.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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TAHRE Is Detected in Different D. melanogaster Stocks
HeT-A and TART are conservative components of Drosophila telomeres. TAHRE was described exclusively in the genome of y1;cn1 bw1 sp1 stock (Abad et al. 2004b
). Screening of genomic libraries of this stock resulted in isolation of 4 TAHRE elements: one is complete and the others are truncated. TAHRE shares sequence similarities with TART (in RT region) as well as with HeT-A (in UTR regions) and may be considered as a result of recombination. In order to specifically detect TAHRE as an independent element by Southern analysis, we prepared 2 PCR-generated TAHRE-specific probes: one (probe 1) is a fragment of RT and the other (probe 2) includes a boundary between the end of the RT ORF and 3' UTR (fig. 1A and Materials and Methods). TART-specific probe (probe 3) is a fragment of TART RT homologous to TAHRE probe 1. Three identical filters were hybridized with probes 1, 2, and 3. The TAHRE-specific probes 1 and 2 reveal identical patterns of genomic DNA restriction fragments (fig. 1B). This means that TAHRE probe 2, in spite of the fact that it contains a fragment of UTR homologous to HeT-A, does not cross-hybridize noticeably to HeT-A. TART-specific probe 3 binds to a distinct pattern of bands. These facts indicate specificity of TAHRE probes.
FISH analysis of the TAHRE probe 1 on polytene chromosomes of the isogenic strain y1;cn1 bw1 sp1 reveals TAHRE in X (2 signals), 2R, and 2L chromosome arms (fig. 1C). This corresponds to the location of TAHRE copies that were previously identified in the genome of this stock by the genomic sequence analysis (Abad et al. 2004b). However, it is not excluded that new TAHRE copies may have appeared in telomeres of this stock in the period from construction of a genomic library in the frame of the Genome Project till now. Thus, the chosen TAHRE probes allow efficient detection of TAHRE elements. FISH analysis reveals TAHRE in some telomeres of D. melanogaster Oregon and Gaiano stocks; no signals were detected in euchromatin and chromocenter (fig. 1C). Thus, TAHRE is a telomeric element; it is present not in all telomeres and is not evenly distributed on telomeres in different D. melanogaster stocks.
We have estimated the number of TAHRE copies in the stocks carrying mutations of Tel-1 and Su(var)2-5 genes. These mutations have been reported to cause telomere lengthening (Savitsky et al. 2002; Siriaco et al. 2002). Mutation in the Su(var)2-5 gene encoding heterochromatic protein 1 causes an increased rate of HeT-A and TART transpositions (Savitsky et al. 2002). Gaiano, a line from the natural population, is considered to be the source of the Tel-1 allele of a putative gene controlling telomere length. Chromosomes of this stock are characterized by long telomeric arrays of HeT-A and TART elements (Siriaco et al. 2002). The product of Tel-1 still has not been identified. To compare the abundance of TAHRE in y1;cn1 bw1 sp1, Gaiano and Su(var)2-504 stocks, semiquantitative PCR analysis of genomic DNA from these stocks was done. Three pairs of TAHRE primers were used in independent experiments (see Materials and Methods and supplementary fig. 1, Supplementary Material online). Loading control was represented by the unique rp49 gene. Figure 2A represents evaluation of TAHRE copy number in genomes of Gaiano and Su(var)2-504 stocks in comparison with that in the y1;cn1 bw1 sp1 genome (Abad et al. 2004b). Telomeric elements are truncated from the 5' end as a result of end underreplication; hence, the number of complete or partial TAHRE elements containing a region complementary to PCR primers used in the analysis was evaluated in our experiments. According to this analysis, Gaiano genomic DNA has twice more TAHRE copies than y1;cn1 bw1 sp1 genome (fig. 2A). Southern analysis using TAHRE probe 1 reveals more intensive and numerous hybridization signals in Gaiano than in y1;cn1 bw1 sp1 stock (fig. 2B). Southern and PCR analyses failed to detect considerable difference in the TAHRE copy number between y1;cn1 bw1 sp1 stock and the stocks containing Su(var)2-504 (fig. 2), Su(var)2-501, Su(var)2-502, and Su(var)2-505 alleles (not shown). FISH analysis of the TAHRE probe on polytene chromosomes of Gaiano detects intensive hybridization signals in X, 2L, 3L, and 3R chromosome arms (fig. 1C). TAHRE elements are more abundant in Gaiano, suggesting that its abundance in a genome is under the control of the Tel-1 locus.
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TAHRE Transposes to Chromosome End
Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, Champion, et al. 1992
; Kahn et al. 2000; Golubovsky et al. 2001), although TART attachments were also detected (Sheen and Levis 1994). Until now, no TAHRE transpositions to chromosome ends have been detected. To study the possibility of TAHRE attachment to chromosome ends, we used truncated X chromosomes with a break in the yellow gene. The break is located in the upstream regulatory region and results in the y2-like phenotype with yellow aristae. Addition of the telomeric retroelement can be monitored by a yellow-to-black change in aristae pigmentation (Savitsky et al. 2002, 2006). Deletion of the yellow locus in the y ac homologue X chromosome a priori obviates the possibility of terminal elongation using a homologous template to perform gene conversion (Kahn et al. 2000; Savitsky et al. 2002). The nature of the attached elements may be determined by PCR amplification and sequencing of the junctions between terminal yellow sequences and retrotransposon attachments. In order to select spontaneous TAHRE attachments, we performed DNA amplification of yellow/telomeric element junction in individuals with changed aristae pigmentation using primers from the yellow gene and TAHRE 3' UTR. Lines with terminal attachments obtained earlier using the same genetic system (Kahn et al. 2000; Savitsky et al. 2002) were also analyzed. In total, genomic DNAs of 57 lines were assayed for the presence of terminal TAHRE attachment; 5 of them were positive for the TAHRE addition to the truncated X chromosome. However, HeT-A and TAHRE are highly homologous in their 3' regions. Therefore, obtained positive results need to be confirmed by cloning of a larger fragment of putative TAHRE attachments. Long-range PCR performed using primers from the yellow gene and TAHRE-specific primer from the RT region yields an amplicon 6 kb in length in 1 of the 5 selected lines. For other lines, we failed to obtain a long-range PCR product, possibly, owing to attachment of truncated elements in these cases. PCR analysis of this product using different sets of TAHRE-specific primers confirms that it is TAHRE element (supplementary fig. 2, Supplementary Material online). This fragment was cloned, sequenced, and aligned to previously described TAHRE elements (Abad et al. 2004b). Sequencing allowed us to identify undoubtedly the attached element as TAHRE. It contains characteristic features of previously described TAHREs including a 3' UTR, similar to HeT-A, adjacent to RT ORF, which is absent in HeT-A elements (fig. 3). This element is practically identical in the coding region and in the 3' UTR with the previously sequenced TAHRE AJ542584 (99.8% identity). It has a 3' oligo(A) stretch, a characteristic feature of recent transposition. 3' UTR of the newly attached TAHRE as well as the earlier described element AJ542584 have a 327-bp deletion compared with other TAHRE copies (AJ542581
[GenBank]
and AJ542582). Thus, the novel telomeric element TAHRE is capable of retrotransposition to chromosome ends.
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The 3' End of TAHRE Has Promoter Activity
HeT-A promoter was earlier found in the 3' end of the element (Danilevskaya et al. 1997
). This promoter drives expression of the downstream element in telomere. We have studied the promoter activity of TAHRE 3' region that is homologous to the HeT-A one using transfection of Drosophila cultured cells by reporter constructs. A total of 400 bp of HeT-A 3' UTR upstream of polyadenylation site were shown to be enough to drive expression of the reporter gene (Danilevskaya et al. 1997). The fragments of HeT-A and TAHRE 3' UTRs were cloned in pCaSpeR-AUG-ß-gal vector containing lacZ reporter gene. TAHRE fragment containing 414 bp upstream of polyadenylation site was derived from the element attached to the broken X chromosome. HeT-A promoter was also subcloned from the element attached to the terminal deleted X chromosome (see Materials and Methods). HeT-A and TAHRE fragments tested for promoter activity in this study have 80% nucleotide identity. The enzyme activity was evaluated in extracts of transfected Schneider 2 cells. We detected a high reporter gene activity of TAHRE constructs, 65 ± 14% of that given by the HeT-A constructs (fig. 4).
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TAHRE-Related Elements in Other Drosophila Species
Using a sequence corresponding to a fragment of D. melanogaster TAHRE RT ORF as a query, TAHRE-related elements were revealed among genomic clones of D. sechellia. CH481023 and CH676464 clones consist of arrays of HeT-A, TART, and TAHRE elements with no other sequences interspersed (supplementary fig. 3, Supplementary Material online). All elements are oriented in a head-to-tail direction, suggesting telomeric origination of these clones. TAHREs located in these clones are characterized by the presence of RT ORF and 3' UTR similar to HeT-A. Independently, TAHRE homologues were identified in the genomes of melanogaster subgroup species by Villasante and colleagues (Villasante et al. 2007, forthcoming).
The presence of TAHRE-related elements in the genomes of a melanogaster subgroup species (Lachaise et al. 2004) was confirmed by Southern blot analysis. A riboprobe corresponding to TAHRE-specific probe 1 (fig. 1A) reveals restriction fragments in the genomic DNA of D. simulans, D. sechellia, D. yakuba, D. mauritiana, D. santomea, and D. erecta (fig. 5A). We saw no hybridization to D. teissieri and D. orena DNAs, possibly, owing to a higher divergence of TAHRE sequences in these species. FISH analysis of the TAHRE probe 1 on polytene chromosomes of D. simulans and D. sechellia salivary glands (fig. 5B and C, respectively) reveals TAHRE in some of the telomeres; no signals were detected in chromocenter. An identical pattern of hybridization was revealed when probe 2 containing a boundary between the end of the RT ORF and 3' UTR was used (not shown).
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Thus, Southern blot, FISH, and genomic analyses data indicate that TAHRE is conserved among the melanogaster subgroup of the Drosophila species telomeric element.
TAHRE Expression in Ovaries of RNAi Mutants
Both HeT-A and TART are targets of the RNAi machinery; however, both elements differ in the character of their response to mutations of RNAi genes. Mutations in spn-E and aub genes, encoding RNA helicase and a protein of the Argonaute protein family, respectively (Gillespie and Berg 1995; Harris and Macdonald 2001; Kennerdell et al. 2002), cause an increase in the TART transcript abundance several times, whereas a strong accumulation of HeT-A transcripts (50 times) is found in homozygous mutants (Savitsky et al. 2006). HeT-A transcripts were previously shown to accumulate in nurse cells and in a growing oocyte in the ovaries of RNAi mutants, whereas TART expression is upregulated in the nurse cells of mutant flies (Vagin et al. 2004; Savitsky et al. 2006).
Abundance of TAHRE transcripts was estimated by RT-PCR and in situ RNA analysis in ovaries of spn-E, aub, vasa, and piwi mutants. The vasa locus contains the vasa gene encoding DEAD-box RNA helicase (Liang et al. 1994) and vig (vasa intronic gene) encoding an RNA-binding protein, a conservative component of RNA-induced silencing complex (Caudy et al. 2002). Piwi as well as Aub belong to the Piwi subfamily of Argonaute protein family, which is involved in germ stem cell maintenance and gametogenesis in different organisms (Cox et al. 1998; Cox et al. 2000; Aravin et al. 2006; Girard et al. 2006; Grivna et al. 2006; Lau et al. 2006).
In situ RNA hybridization using a TAHRE-specific probe containing an RT fragment was performed. TAHRE sense transcripts are detected in growing oocytes and in the cytoplasm of nurse cells in ovaries of spn-E1/spn-Ehls3987, piwi2/piwi3, and vigEP812/vasPH165 heteroallelic flies and in nurse cells at the late stages of oogenesis in ovaries of aubQC42/aubHN females (fig. 6A). Such distribution of TAHRE transcripts resembles a pattern of HeT-A transcript accumulation in RNAi mutants rather than TART expression upregulated only in the nurse cells (Vagin et al. 2004; Savitsky et al. 2006). Figure 6B demonstrates a difference in the expression pattern of TART, HeT-A, and TAHRE and a similarity of HeT-A and TAHRE transcript distribution in ovaries of piwi mutants. TAHRE antisense transcripts were not detected in ovaries of RNAi mutants by in situ RNA hybridization (not shown).
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The abundance of TAHRE transcripts was compared by RT-PCR using oligo(dT) primer in ovaries of heterozygous and heteroallelic RNAi mutants. TAHRE-specific primers from RT-coding region were used. Because the level of antisense TAHRE transcripts detected by in situ RNA hybridization is not affected in RNAi mutants, the observed changes can be attributed to the abundance of sense TAHRE transcripts. We observed a drastic accumulation of TAHRE transcripts in ovaries of heteroallelic spn-E1/spn-Ehls3987, aubQC42/aubHN, vigEP812/vasPH165, and piwi2/piwi3 flies (fig. 6C). A strong derepression (more than 10 times) of TAHRE in RNAi mutants is similar to a HeT-A transcript accumulation in mutants, opposed to the dosage effect of the same mutations on TART expression (Savitsky et al. 2006
). Both VIG and VASA proteins are absent in the ovaries of homozygous vigEP812 and vasPH165 mutants (not shown). For the present moment, it is unclear whether VIG or VASA are involved in the silencing of TAHRE element.
Mutations in spn-E eliminate HeT-A– and TART-specific short RNAs in the germ line (Savitsky et al. 2006). We further tested for the presence of short RNA species homologous to TAHRE in ovary RNA isolated from spn-E mutants. As a control, the isogenic strain y1;cn1 bw1 sp1 was used. Northern hybridization using TAHRE-specific sense probe to detect antisense transcripts reveals the presence of heterogeneous 25–29 nt RNA species in the ovarian RNA isolated from y1;cn1 bw1 sp1 and heterozygous spn-E1/+ flies (fig. 6D). No TAHRE short RNAs were detected in RNA from heteroallelic spn-E1/spn-Ehls3987 ovaries. Expression of micro RNA (mir-13b1) in ovaries is not affected by spn-E mutation and was used as a loading control (fig. 6D). Our results show a negative correlation between the expression of the TAHRE element and the presence of a short RNA guiding dsRNA-mediated silencing.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Drosophila telomeres are maintained by successive transpositions of specialized telomeric retroelements HeT-A, TART, and, as shown here, TAHRE. A novel telomeric element, TAHRE was identified in silico among telomere-specific clones of the y1;cn1 bw1 sp1 strain of D. melanogaster (Abad et al. 2004b
). All described TAHRE elements possessed such characteristic peculiarities of a novel family as the presence of the RT ORF and 3' UTR similar to the HeT-A 3' UTR. Identification of TAHRE allowed the authors to propose an evolutionary origin of HeT-A and TART from a common ancestor (Abad et al. 2004b).
Our studies have shown the presence of TAHRE elements in all investigated D. melanogaster stocks as well as in other Drosophila species. TAHRE was found in D. sechellia genomic clones among arrays of other telomeric elements. Southern analysis reveals TAHRE in the genomic DNA of the majority of the species of the melanogaster subgroup. Thus, TAHRE is a conserved telomeric retrotransposon present in Drosophila at least 10–15 MYA, the time of divergence of melanogaster subgroup species from a common ancestor (Lachaise et al. 2004). De novo attachment of TAHRE to chromosome end indicates that this element is an equal counterpart of telomere maintenance in Drosophila. A high sequence similarity of TAHRE in different D. melanogaster stocks (99.8%) may result from its conservative role in telomere maintenance. It was shown earlier that TART-A element from y1;cn1 bw1 sp1 stock is 99.3% identical to a TART-A from the distantly related Oregon R stock (Abad et al. 2004a; George et al. 2006). These data suggest an important conservative function for TART and TAHRE in telomere maintenance. Of course, the structural role is not a primary function for TART and TAHRE as no TART and TAHRE elements are detected in some telomeres, these elements are not evenly distributed on telomeres (Levis et al. 1993; Abad et al. 2004a; present study). One of the reasons of a long cooperation of several telomeric elements throughout evolution may be the distribution of different roles among elements.
The main structural component of telomeres is the HeT-A family elements. All of the D. melanogaster stocks analyzed have both HeT-A and TART elements, and the relative copy number of HeT-A and TART across stock lines is characterized by a proportional relationship and amounts to around 30 HeT-A and 10 TART copies (Abad et al. 2004a; George et al. 2006). A single complete TAHRE copy was identified in y1;cn1 bw1 sp1 telomeres (Abad et al. 2004b). Retroelement HeT-A lacks a gene encoding RT, although this activity is obviously supplied in trans from an unknown source because retrotransposition of HeT-A to chromosome ends is the main mechanism of telomere elongation. According to the cis preference rule, retrotransposon RT preferentially binds RNA of the element that encodes it (Wei et al. 2001). In this case, TART and TAHRE might be expected to be the most abundant telomere elements. However, only HeT-A Gag associates with telomeres, suggesting a potential targeting of the HeT-A mRNA to chromosome ends (Rashkova et al. 2002, 2003). Evidently, telomere targeting of retrotransposon mRNA is the most crucial stage in telomere elongation and HeT-A Gag gives an advantage to HeT-A mRNA in this process. The unique complete TAHRE copy located on the X chromosome was proposed to be a master copy that supplies the RT activity controlling HeT-A transpositions (Abad et al. 2004b). TART may also be considered as a source of RT for HeT-A as well as for its own transpositions (Rashkova et al. 2002). However, TART and HeT-A, in spite of sharing the region of integration, are not similar in the pattern of their expression (Danilevskaya et al. 1999; Savitsky et al. 2006). In the germ line, sense and antisense TART transcripts accumulate in nurse cells predominantly at the late stages of oogenesis, whereas HeT-A transcripts are detected in a growing oocyte from the earlier stages of oogenesis. The present study has revealed a similar distribution of TAHRE and HeT-A transcripts in the germ line, namely, TAHRE and HeT-A transcripts, unlike TART, accumulate in an oocyte, where, in fact, terminal transpositions occur. This fact implies TAHRE rather than TART as a source of RT for HeT-A transpositions. HeT-A and TAHRE sequence similarity may be considered as a cause of a common pattern of their transcripts distribution. Actually, highly homologous RNA-binding Gag proteins and similar 3' UTRs may be responsible for a similar HeT-A and TAHRE transcript distribution in a cell and facilitate functional cooperation of the elements. On the other hand, this resemblance may be determined by a common promoter. HeT-A promoter is located at the end of its 3' UTR and drives transcription of the downstream element (Danilevskaya et al. 1997). The 3' ends of HeT-A elements were located upstream of the 4 cloned TAHRE elements (Abad et al. 2004b). TAHRE read-through transcription from HeT-A promoter was demonstrated (Frydrychova et al. 2007). Peculiarities of a promoter activity may be responsible for cellular distribution of transcripts. Actually, 500 bp of the HeT-A 3' UTR are capable of driving a reporter gene expression in the endogenous HeT-A stage- and cell-type–specific manner (George and Pardue 2003). In any case, sequence similarity of HeT-A and TAHRE 3' UTRs and a common pattern of their expression allow us to consider TAHRE as a source of RT enzymatic activity for HeT-A transpositions in ovaries. Interestingly, the 3' end of TAHRE, as well as HeT-A element, comprises a promoter activity. However, in contrast to widely presented HeT-A elements, TAHRE is a minor component of telomeres and its 3' promoter is not necessary for the expression of other TAHRE elements. Possibly, conservation of TAHRE 3' UTR promoter activity reflects that this element was a major telomeric element in the evolutionary recent past.
Both HeT-A and TART were shown to be targets of the RNAi system, although their response to RNAi mutations was different (Savitsky et al. 2006). Mutations in spn-E or aub RNAi genes caused a 2- or 4-fold increase in the TART transcript abundance in a dosage-dependent manner. A strong accumulation of HeT-A transcripts (50 times) was found in ovaries of homozygous RNAi mutants. We observed a drastic accumulation of TAHRE transcripts (more than 10 times) in ovaries of RNAi mutants. This effect is similar to the HeT-A transcript accumulation in RNAi mutants (Vagin et al. 2004; Savitsky et al. 2006).
spn-E and aub mutations in the heterozygous state considerably increased TART transpositions to the broken chromosome end. No other attachments to a terminal deleted chromosome besides TART were detected in spn-Ehls3987 and aubQC42 mutant alleles (Savitsky et al. 2006). In spite of the fact that we have not monitored the TAHRE attachments, it is obvious that HeT-A as well as TAHRE transpositions are not considerably affected by one dosage of RNAi mutant genes. In all these cases, TAHRE resembles HeT-A more than the TART element.
Derepression of TAHRE expression in ovaries of RNAi mutants argues that TAHRE as well as HeT-A and TART is a target of the RNAi machinery in the germ line. Small RNAs produced from the telomeric element TAHRE belong to a long size class of fly repeat-associated small interfering RNAs (rasiRNAs) (25–29 nt). In contrast to 21–22 nt siRNAs guiding posttranscriptional RNAi (Elbashir et al. 2001), rasiRNAs are involved in a distinct RNAi-based pathway (Vagin et al. 2006). Silencing of TAHRE requires a putative RNA helicase Spn-E and Piwi subfamily Argonaute proteins Piwi and Aub. These proteins are the components of a fly rasiRNA pathway involved in the silencing of the repetitive elements such as retrotransposons and endogenous repeats (Aravin et al. 2001; Vagin et al. 2004; Kalmykova et al. 2005; Vagin et al. 2006). It remains to be determined, however, which product of vasa locus, RNA helicase VASA or RNA-binding protein VIG, is involved in the silencing of telomeric elements. Telomeric retroelements, in spite of their vital cellular function, are also the targets of a rasiRNA-silencing pathway (Vagin et al. 2004; Savitsky et al. 2006; Vagin et al. 2006). One distinct feature of this RNA-silencing pathway is that it functions in the germ line. Thus, telomere elongation occurs at premeiotic stages of oogenesis in gamete precursors under a negative control of the rasiRNA-mediated silencing mechanism (Savitsky et al. 2006). Do rasiRNAs of telomeric elements guide posttranscriptional degradation of their mRNA or are they involved in formation of telomeric chromatin? Are HeT-A, TART, and TAHRE rasiRNA pathways functionally different in the context of telomere functioning? Is the presence of the 3 families of telomeric elements at Drosophila telomeres redundant, or does each element play a distinct role? The answer to these questions will help us to elucidate how retrotransposons, RNAi system, and host genome coevolve to provide Drosophila telomere homeostasis.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary materials including figure 1 (TAHRE copy number), figure 2 (TAHRE terminal attachment), and figure 3 (TAHRE in D. sechellia telomeres) are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank V. Gvozdev and A. Villasante for critical reading of the manuscript and V. Vagin, E. Pasyukova, and S. Lavrov for helpful methodological advices. This work was supported by Russian Academy of Sciences program for Molecular and Cell Biology to A.K. and M.S., grant from the Russian Foundation for Basic Researches (06-04-48493), International Research Scholar Award from the Howard Hughes Medical Institute (to P.G.), and a stipend from the Center for Medical Studies, University of Oslo to M.S.
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1 S.S. and D.K. equally contributed to this study.
Koichiro Tamura, Associate Editor
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|---|
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
|---|
Abad JP, de Pablos B, Osoegawa K, de Jong PJ, Martin-Gallardo A, Villasante A. Genomic analysis of Drosophila melanogaster telomeres: full-length copies of HeT-A and TART elements at telomeres. Mol Biol Evol (2004a) 21:1613–1619.
Abad JP, de Pablos B, Osoegawa K, de Jong PJ, Martin-Gallardo A, Villasante A. TAHRE, a novel telomeric retrotransposon from Drosophila melanogaster, reveals the origin of Drosophila telomeres. Mol Biol Evol (2004b) 21:1620–1624.
Aravin A, Gaidatzis D, Pfeffer S, et al, 17 co-authors. A novel class of small RNAs bind to MILI protein in mouse testes. Nature (2006) 442:203–207.[Medline]
Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM, Gvozdev VA. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr Biol (2001) 11:1017–1027.[CrossRef][Web of Science][Medline]
Arkhipova IR, Morrison HG. Three retrotransposon families in the genome of Giardia lamblia: two telomeric, one dead. Proc Natl Acad Sci USA (2001) 98:14497–14502.
Ashburner M. Drosophila: a laboratory manual (1989) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Biessmann H, Champion LE, O'Hair M, Ikenaga K, Kasravi B, Mason JM. Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends. EMBO J (1992) 11:4459–4469.[Web of Science][Medline]
Biessmann H, Valgeirsdottir K, Lofsky A, Chin C, Ginther B, Levis RW, Pardue ML. HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster. Mol Cell Biol (1992) 12:3910–3918.
Bryan TM, Cech TR. Telomerase and the maintenance of chromosome ends. Curr Opin Cell Biol (1999) 11:318–324.[CrossRef][Web of Science][Medline]
Casacuberta E, Pardue M.-L. Coevolution of the telomeric retrotransposons across Drosophila species. Genetics (2002) 161:1113–1124.
Casacuberta E, Pardue M.-L. HeT-A elements in Drosophila virilis: retrotransposon telomeres are conserved across the Drosophila genus. Proc Natl Acad Sci USA (2003a) 100:14091–14096.
Casacuberta E, Pardue M-L. Transposon telomeres are widely distributed in the Drosophila genus: TART elements in the virilis group. Proc Natl Acad Sci USA (2003b) 100:3363–3368.
Caudy AA, Myers M, Hannon GJ, Hammond SM. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev (2002) 16:2491–2496.
Cenci G, Ciapponi L, Gatti M. The mechanism of telomere protection: a comparison between Drosophila and humans. Chromosoma (2005) 114:135–145.[CrossRef][Web of Science][Medline]
Cooper JP. Telomere transitions in yeast: the end of the chromosome as we know it. Curr Opin Genet Dev (2000) 10:169–177.[CrossRef][Web of Science][Medline]
Cox DN, Chao A, Baker J, Chang L, Qiai D, Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev (1998) 12:3715–3727.
Cox DN, Chao A, Lin H. piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development (2000) 127:503–514.[Abstract]
Danilevskaya ON, Arkhipova IR, Traverse KL, Pardue ML. Promoting in tandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell (1997) 88:647–655.[CrossRef][Web of Science][Medline]
Danilevskaya ON, Lowenhaupt K, Pardue ML. Conserved subfamilies of the Drosophila HeT-A telomere-specific retrotransposon. Genetics (1998) 148:233–242.
Danilevskaya ON, Tan C, Wong CJ, Alibhai M, Pardue ML. Unusual features of the Drosophila melanogaster telomere transposable element HeT-A are conserved in D. yakuba telomere elements. Proc Natl Acad Sci USA (1998) 95:3770–3775.
Danilevskaya ON, Traverse KL, Hogan NC, DeBaryshe PG, Pardue ML. The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol (1999) 19:873–881.
Eickbush TH. Telomerase and retrotransposons: which came first? Science (1997) 277:911–912.
Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev (2001) 15:188–200.
Frydrychova RC, Biessmann H, Konev AY, Golubovsky MD, Johnson J, Archer TK, Mason JM. Transcriptional activity of the telomeric retrotransposon HeT-A in Drosophila melanogaster is stimulated as a consequence of subterminal deficiencies at homologous and nonhomologous telomeres. Mol Cell Biol (2007) 27:4991–5001.
George JA, DeBaryshe PG, Traverse KL, Celniker SE, Pardue ML. Genomic organization of the Drosophila telomere retrotransposable elements. Genome Res (2006) 316:1231–1240.
George JA, Pardue ML. The promoter of the heterochromatic Drosophila telomeric retrotransposon, HeT-A, is active when moved into euchromatic locations. Genetics (2003) 163:625–635.
Gillespie DE, Berg CA. Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases. Genes Dev (1995) 9:2495–2508.
Girard A, Sachidanandam R, Hannon GJ, Carmell MA. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature (2006) 442:199–202.[Medline]
Golubovsky MD, Konev AY, Walter MF, Biessmann H, Mason JM. Terminal retrotransposons activate a subtelomeric white transgene at the 2L telomere in Drosophila. Genetics (2001) 158:1111–1123.
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell (1985) 43:405–413.[CrossRef][Web of Science][Medline]
Grivna ST, Beyret E, Wang Z, Lin H. A novel class of small RNAs in mouse spermatogenic cells. Genes Dev (2006) 20:1709–1714.
Harris AN, Macdonald PM. Aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. Development (2001) 128:2823–2832.[Web of Science][Medline]
Higashiyama T, Noutoshi Y, Fujie M, Yamada T. Zepp, a LINE-like retrotransposon accumulated in the Chlorella telomeric region. EMBO J (1997) 16:3715–3723.[CrossRef][Web of Science][Medline]
Kahn T, Savitsky M, Georgiev P. Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms. Mol Cell Biol (2000) 20:7634–7642.
Kalmykova AI, Klenov MS, Gvozdev VA. Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res (2005) 33:2052–2059.
Kalmykova AI, Kwon DA, Rozovsky YM, Hueber N, Capy P, Maisonhaute C, Gvozdev VA. Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes. Mol Biol Evol (2004) 21:2281–2289.
Kennerdell JR, Yamaguchi S, Carthew RW. RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E. Genes Dev (2002) 16:1884–1889.
Kogan GL, Tulin AV, Aravin AA, Abramov YA, Kalmykova AI, Maisonhaute C, Gvozdev VA. The GATE retrotransposon in Drosophila melanogaster: mobility in heterochromatin and aspects of its expression in germline tissues. Mol Genet Genomics (2003) 269:234–242.[CrossRef][Web of Science][Medline]
Lachaise D, Capy P, Cariou M-L, Joly D, Lemeunier F, David JR. Nine relatives from one African ancestor: population biology and evolution of the Drosophila melanogaster subgroup species. In: The evolution of population biology—Singh RS, Uyenoyama MK, eds. (2004) Cambridge, UK: Cambridge University Press. 315–343.
Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP, Kingston RE. Characterization of the piRNA complex from rat testes. Science (2006) 313:363–367.
Lavrov S, Dejardin J, Cavalli G. Combined immunostaining and FISH analysis of polytene chromosomes. Methods Mol Biol (2004) 247:289–303.[Medline]
Levis RW, Ganesan R, Houtchens K, Tolar LA, Sheen FM. Transposons in place of telomeric repeats at a Drosophila telomere. Cell (1993) 75:1083–1093.[CrossRef][Web of Science][Medline]
Liang L, Diehl-Jones W, Lasko P. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development (1994) 120:1201–1211.[Abstract]
Mikhailovsky S, Belenkaya T, Georgiev P. Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster. Chromosoma (1999) 108:114–120.[CrossRef][Web of Science][Medline]
Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB, Cech TR. Telomerase catalytic subunit homologues from fission yeast and human. Science (1997) 277:955–959.
Okazaki S, Ishikawa H, Fujiwara H. Structural analysis of TRAS1, a novel family of telomeric repeat-associated retrotransposons in the silkworm, Bombyx mori. Mol Cell Biol (1995) 15:4545–4552.[Abstract]
Rashkova S, Athanasiadis A, Pardue ML. Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons. J Virol (2003) 77:6376–6384.
Rashkova S, Karam SE, Kellum R, Pardue ML. Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends. J Cell Biol (2002) 159:397–402.
Savitsky M, Kravchuk O, Melnikova L, Georgiev P. Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol Cell Biol (2002) 22:3204–3218.
Savitsky M, Kwon D, Georgiev P, Kalmykova A, Gvozdev V. Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline. Genes Dev (2006) 20:345–354.
Sheen FM, Levis RW. Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc Natl Acad Sci USA (1994) 91:12510–12514.
Shi H, Djikeng A, Tschudi C, Ullu E. Argonaute protein in the early divergent eukaryote Trypanosoma brucei: control of small interfering RNA accumulation and retroposon transcript abundance. Mol Cell Biol (2004) 24:420–427.
Sijen T, Plasterk RH. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature (2003) 426:310–314.[CrossRef][Medline]
Siriaco GM, Cenci G, Haoudi A, Champion LE, Zhou C, Gatti M, Mason JM. Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres. Genetics (2002) 160:235–245.
Styhler S, Nakamura A, Swan A, Suter B, Lasko P. vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development (1998) 125:1569–1578.[Abstract]
Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, Schultz RM. RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev Biol (2004) 269:276–285.[CrossRef][Web of Science][Medline]
Takahashi H, Okazaki S, Fujiwara H. A new family of site-specific retrotransposons, SART1, is inserted into telomeric repeats of the silkworm, Bombyx mori. Nucleic Acids Res (1997) 25:1578–1584.
Vagin VV, Klenov MS, Kalmykova AI, Stolyarenko AD, Kotelnikov RN, Gvozdev VA. The RNA interference proteins and vasa locus are involved in the silencing of retrotransposons in the female germline of Drosophila melanogaster. RNA Biol (2004) 1:54–58.[Medline]
Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD. A distinct small RNA pathway silences selfish genetic elements in the germline. Science (2006) 313:320–324.
Villasante A, Abad JP, Planello R, Mendez-Lago M, Celniker SE, de Pablos B, Forthcoming. Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase. Genome Research (2007).
Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, Kazazian HH, Boeke JD, Moran JV. Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol (2001) 21:1429–1439.
Wu-Scharf D, Jeong B, Zhang C, Cerutti H. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science (2000) 290:1159–1162.
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