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• W2071916965 abstract "Non-long terminal repeat retrotransposons, widespread among eukaryotic genomes, transpose by reverse transcription of an RNA intermediate. Some of them, like L1 in the human, terminate at the 3′-end with a poly(dA) stretch whereas others, like the I factor in Drosophila melanogaster, have instead a short sequence repeated in tandem. This suggests different requirements for the initiation of reverse transcription. Here, we have used an RNA circularization/reverse transcription-PCR technique to analyze the 5′- and 3′-ends of the full-length transcripts produced by the I factor at the time of active retrotransposition. These transcripts are capped and polyadenylated similar to conventional messenger RNAs. We have analyzed the 3′-ends of transcripts and transposed copies produced by I elements mutated at the 3′-ends. Transcripts devoid of tandem UAA repeats, although capable of building the components of the retrotransposition machinery, are inefficiently used as retrotransposition intermediates. Such transcripts produce rare new integrated copies issued from the inaccurate initiation of reverse transcription near the 3′-end of the element. The tandem UAA repeats at the 3′-end of the transcripts of I are required for the efficient and precise initiation of reverse transcription. This strong specificity of the I factor reverse transcriptase for its own transcript has implications for the impact of I factor retrotransposition on the host genome. Non-long terminal repeat retrotransposons, widespread among eukaryotic genomes, transpose by reverse transcription of an RNA intermediate. Some of them, like L1 in the human, terminate at the 3′-end with a poly(dA) stretch whereas others, like the I factor in Drosophila melanogaster, have instead a short sequence repeated in tandem. This suggests different requirements for the initiation of reverse transcription. Here, we have used an RNA circularization/reverse transcription-PCR technique to analyze the 5′- and 3′-ends of the full-length transcripts produced by the I factor at the time of active retrotransposition. These transcripts are capped and polyadenylated similar to conventional messenger RNAs. We have analyzed the 3′-ends of transcripts and transposed copies produced by I elements mutated at the 3′-ends. Transcripts devoid of tandem UAA repeats, although capable of building the components of the retrotransposition machinery, are inefficiently used as retrotransposition intermediates. Such transcripts produce rare new integrated copies issued from the inaccurate initiation of reverse transcription near the 3′-end of the element. The tandem UAA repeats at the 3′-end of the transcripts of I are required for the efficient and precise initiation of reverse transcription. This strong specificity of the I factor reverse transcriptase for its own transcript has implications for the impact of I factor retrotransposition on the host genome. Very little is known about the mechanism of retrotransposition of non-LTR 1The abbreviations used are: non-LTRnon-long terminal repeatORFopen reading frameUTRuntranslated regionRTreverse transcriptionTKthymidine kinaseChaCharolles reactive strain elements that are widespread among eukaryotic genomes. Most current knowledge comes from in vitro studies of the site-specific R2 elements from Bombyx mori. These studies indicate that reverse transcription of a full-length RNA intermediate of transposition occurs at the site of integration, using a 3′-hydroxyl group generated by endonucleolytic cleavage of the genomic DNA to prime synthesis of the first cDNA strand (1Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Cell. 1993; 72: 595-605Abstract Full Text PDF PubMed Scopus (932) Google Scholar). This target-primed reverse transcription process is mediated by endonuclease and reverse transcriptase activities encoded by the single open reading frame (ORF) of R2 elements (1Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Cell. 1993; 72: 595-605Abstract Full Text PDF PubMed Scopus (932) Google Scholar, 2Yang J. Eickbush T.H. Mol. Cell. Biol. 1998; 18: 3455-3465Crossref PubMed Scopus (33) Google Scholar, 3Xiong Y. Eickbush T.H. Mol. Biol. Evol. 1988; 5: 675-690PubMed Google Scholar). Many non-LTR retrotransposons, including L1 in the human and the I factor in Drosophila, differ from R2 in that they possess an additional ORF encoding a protein probably involved in ribonucleoparticle formation (4Dawson A. Hartswood E. Paterson T. Finnegan D.J. EMBO J. 1997; 16: 4448-4455Crossref PubMed Scopus (53) Google Scholar, 5Hohjoh H. Singer M.F. EMBO J. 1996; 15: 630-639Crossref PubMed Scopus (292) Google Scholar, 6Pont-Kingdon G. Chi E. Christensen S. Carroll D. Nucleic Acids Res. 1997; 25: 3088-3094Crossref PubMed Scopus (16) Google Scholar) and maybe also in reverse transcription (7Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Crossref PubMed Scopus (288) Google Scholar). In addition, their endonucleases have similarities with apurinic/apyrimidinic endonucleases (8Martin F. Maranon C. Olivares M. Alonso C. Lopez M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (127) Google Scholar, 9Martin F. Olivares M. Lopez M.C. Alonso C. Trends Biochem. Sci. 1996; 21: 283-285Abstract Full Text PDF PubMed Google Scholar), whereas the endonuclease of R2 elements is related to bacterial restriction endonucleases (10Yang J. Malik H.S. Eickbush T.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7847-7852Crossref PubMed Scopus (130) Google Scholar). Nevertheless, although direct experimental evidence is lacking, many observations are consistent with the idea that other non-LTR elements also use the target-primed reverse transcription mechanism of retrotransposition (1Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Cell. 1993; 72: 595-605Abstract Full Text PDF PubMed Scopus (932) Google Scholar, 11Ostertag E.M. Kazazian H.H., Jr. Annu. Rev. Genet. 2001; 35: 501-538Crossref PubMed Scopus (628) Google Scholar). The 3′-end of the RNA intermediate of transposition is crucial in this mechanism because it contains the site of initiation of reverse transcription. Studies of human L1 indicate that the poly(A) tail at the 3′-end of the transcript is used as the reverse transcription initiation site rather than specific sequences in the 3′-UTR of the element (12Moran J.V. DeBerardinis R.J. Kazazian H.H., Jr. Science. 1999; 283: 1530-1534Crossref PubMed Scopus (485) Google Scholar, 13Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian H.H., Jr. Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). As a result, transposed copies of L1 terminate with a poly(dA) stretch at the 3′-end. Some non-LTR retrotransposons, such as the I factor in Drosophila melanogaster, do not terminate with a poly(dA) stretch but rather with a short sequence repeated in tandem, suggesting the use of an alternative mode of the initiation of reverse transcription. non-long terminal repeat open reading frame untranslated region reverse transcription thymidine kinase Charolles reactive strain The I factor is a non-LTR retrotransposon of particular interest because its transposition occurs at high frequencies in “Inducer-Reactive” hybrid dysgenic females (14Picard G. Bucheton A. Lavige J.M. Pelisson A. C. R. Acad. Sci. (Paris). 1976; 282: 1813-1816Google Scholar) (for a recent review see Ref. 15Bucheton A. Busseau I. Teninges D. Craig N.L. Craigie R. Gellert M. Lambowitz A. Mobile DNA II. ASM Press, Washington D. C.2002: 796-812Crossref Scopus (4) Google Scholar), allowing in vivo analysis of the mechanism of retrotransposition. Transposition of I factors occurs at high frequencies in the germ line of hybrid females, called SF females, produced by crosses between females from reactive strains that lack functional I factors and males from inducer strains containing active I factors (14Picard G. Bucheton A. Lavige J.M. Pelisson A. C. R. Acad. Sci. (Paris). 1976; 282: 1813-1816Google Scholar, 16Bucheton A. Paro R. Sang H.M. Pelisson A. Finnegan D.J. Cell. 1984; 38: 153-163Abstract Full Text PDF PubMed Scopus (188) Google Scholar). Transposition is accompanied by a characteristic syndrome of sterility; some of the embryos produced by SF females die early in development. The degree of sterility (the proportion of the embryos that die) correlates with the frequency of transposition (17Picard G. Mol. Gen. Genet. 1978; 164: 235-247Crossref Scopus (32) Google Scholar). Active I factors possess all the typical features of non-LTR retrotransposons. They contain two ORFs. ORF1 encodes a nucleic acid-binding protein containing cysteine-rich motifs (4Dawson A. Hartswood E. Paterson T. Finnegan D.J. EMBO J. 1997; 16: 4448-4455Crossref PubMed Scopus (53) Google Scholar). ORF2 encodes a putative polypeptide with endonuclease, reverse transcriptase, and RNase H domains (8Martin F. Maranon C. Olivares M. Alonso C. Lopez M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (127) Google Scholar, 18Feng Q. Moran J.V. Kazazian H.H., Jr. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar, 19Fawcett D.H. Lister C.K. Kellett E. Finnegan D.J. Cell. 1986; 47: 1007-1015Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 20Abad P. Vaury C. Pelisson A. Chaboissier M.C. Busseau I. Bucheton A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8887-8891Crossref PubMed Scopus (48) Google Scholar). I factors terminate at the 3′-ends by several (3Xiong Y. Eickbush T.H. Mol. Biol. Evol. 1988; 5: 675-690PubMed Google Scholar, 4Dawson A. Hartswood E. Paterson T. Finnegan D.J. EMBO J. 1997; 16: 4448-4455Crossref PubMed Scopus (53) Google Scholar, 5Hohjoh H. Singer M.F. EMBO J. 1996; 15: 630-639Crossref PubMed Scopus (292) Google Scholar, 6Pont-Kingdon G. Chi E. Christensen S. Carroll D. Nucleic Acids Res. 1997; 25: 3088-3094Crossref PubMed Scopus (16) Google Scholar, 7Martin S.L. Bushman F.D. Mol. Cell. Biol. 2001; 21: 467-475Crossref PubMed Scopus (288) Google Scholar, 8Martin F. Maranon C. Olivares M. Alonso C. Lopez M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (127) Google Scholar) repeats of a TAA triplet instead of the poly(A) stretches found in many other non-LTR retrotransposons and are flanked by target site duplications of variable lengths. The transcription of I factors is initiated from an internal RNA polymerase II promoter lying within the 5′-UTR (21McLean C. Bucheton A. Finnegan D.J. Mol. Cell. Biol. 1993; 13: 1042-1050Crossref PubMed Scopus (85) Google Scholar). It produces a full-length 5.4-kb transcript, the abundance of which correlates with the transposition frequency (22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar). This 5.4-kb transcript is believed to serve both as a bicistronic messenger for the synthesis of the products of the two open reading frames and as the retrotransposition intermediate (22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar, 23Bouhidel K. Terzian C. Pinon H. Nucleic Acids Res. 1994; 22: 2370-2374Crossref PubMed Scopus (23) Google Scholar). We have used a technique that relies on RNA circularization followed by RT-PCR to characterize in detail the structures of the 5′- and 3′-ends of full-length transcripts produced by actively transposing I factors. These transcripts follow the classical messenger RNA maturation pathway. The study of transcripts and transposed copies produced by elements modified at the 3′-ends indicate that the tandem TAA repeats at the 3′-end of the I factor are essential for efficient and precise retrotransposition. The pI954 construct has the I954 element inserted into the transformation vector pUChsneo (24Pritchard M.A. Dura J.M. Pelisson A. Bucheton A. Finnegan D.J. Mol Gen. Genet. 1988; 214: 533-540Crossref PubMed Scopus (40) Google Scholar). To generate ITK and IΔ3TK, the 3′-end of I954 was amplified using primers 3′1 (5′-ACGGATCCGTCCATGGTACCAATC-3′) and 3′4 (5′-TAACCCGGGTTATTATTATTATTATGATAGATAGAATAG-3′) and primers 3′1 (5′-ACGGATCCGTCCATGGTACCAATC-3′) and 3′3 (5′-TAACCCGGGATGATAGATAGAATAGTTTACAAAAC-3′), respectively. These PCR products were cloned into the BamHI/XmaI-cut plasmid pBTK2 (a kind gift from Aude Le Roux) that contains the TK gene of the herpes simplex virus. Acc65I/EcoRI fragments from these constructs were used to replace the Acc65I/EcoRI fragment of pI954 containing the 3′-end of I. To generate IΔ3CC, the 3′-end of I954 was amplified with primers 3′TCC (5′-TTGAATTCGGATGATAGATAGAATAGTTTAC-3′) and Cl3 (5′-CTGCAGTCCATGGTACAATCTATTAAC-3′), digested with Acc65I/EcoRI, and used to replace the Acc65I/EcoRI fragment of pI954. P-mediated transformation of the wild type reactive strain Cha (Charolles) was performed as described (25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar). Typically, 10–13 Cha females were mated to 8–10 transgenic males on fresh medium and transferred on new medium the next day. Eggs were left to develop for 24 h at 25 °C, and the percentage of hatched eggs was determined. Only samples of more than 100 embryos were taken into account. Mean values were calculated from at last three independent crosses for each transgenic line. 20 units of recombinant RNasin ribonuclease inhibitor (Promega) were added to all reactions involving RNA. Total RNA was extracted from 50 pairs of ovaries with the RNeasy® Midi kit (Qiagen). 50 μg of RNA were treated with 20 units of RNase-free DNase I (Promega) in 100 μl for 1 h at 37 °C. After phenol/chloroform extractions, the RNAs were ethanol precipitated with 2.5 m ammonium acetate and resuspended in 20 μl of H2O. For intramolecular ligation, 20 μg of RNA were incubated with 2.5 units of tobacco acid pyrophosphatase (Epicentre Technologies) in 20 μl for 1 h at 37 °C. After ethanol precipitation in 2.5 mammonium acetate, 5 μg of RNAs were incubated with 20 units of T4 RNA ligase (Epicentre Technologies) in 400 μl of optimal buffer (50 mm Tris HCl, pH 7.5, 10 mm MgCl2, 20 mm dithiothreitol, 100 μm ATP, and 100 μg/ml acetylated bovine serum albumin) for 16 h at 16 °C. After phenol/chloroform extraction, the ligated RNA was ethanol precipitated in 2.5 m ammonium acetate. 400 ng of RNAs were reverse transcribed using the ThermoScript™ RT-PCR System (Invitrogen) and 10 pmol of primer 2cir5′ (5′-CATCCCTCAACTTCTCCTCC-3′) for 1 h at 59 °C. The sample was then treated for 20 min at 37 °C with 2 units of RNase H. Nested PCR amplifications were performed on 2 μl of the RT-PCR reaction, first with primers B290 (5′-TCGAAAGAGTTGTTGTC-3′) and Ri160 (5′-GTACATAACAAGCCAGCAATTAG-3′) and second with primers B95 (5′-GATTTTGCTGATAAGAG-3′) and 2cir3′up (5′-CCCCGTAGCTAATGCTATACTATC-3′). The PCR products were cloned using the TOPO TA Cloning® Kit (Invitrogen) and sequenced by Genome Express. The positions of the oligonucleotides at the 5′- and 3′-ends of the I954 element are shown in Fig. 1a. 5 μg of RNA were reverse transcribed with 20 pmol of anchor-oligo(dT) primer Ad1dT (5′-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3′) for 1 h at 52 °C using the ThermoScript™ RT-PCR System (Invitrogen). Two sequential PCR amplifications were performed, first with Cl3 and Ad1 (5′-GCGAGCTCCGCGGCCGCG-3′) and second with sev3′up (5′-CTTTAAACCACATATTTAACTCATG-3′) and Ad1 (Fig. 1a). The PCR products were electroeluted from a 5% polyacrylamide gel, cloned, and sequenced as above. Genomic DNA was extracted from pools of 25 males using standard procedures. Inverse PCR experiments were performed as described by Chaboissier et al. (25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar), except that the restriction enzyme MboI was used for digestion, and oligonucleotides 7 (5′-TCGCAAGGTCGGCTTTAAGG-3′) and 3 (5′-ACCCTCTAGACCTTCTTAGC-3′) were used in PCR amplifications. PCR products were cloned using the Stratagene PCR-Script™ Amp cloning kit and sequenced by Genome Express. RNA detection by in situ hybridization on whole mount ovaries was performed following the protocol of Tautz and Pfeifle (26Tautz D. Pfeifle C. Chromosoma (Berlin). 1989; 98: 81-85Crossref PubMed Scopus (2090) Google Scholar) adapted by Capri et al. (27Capri M. Santoni M.J. Thomas-Delaage M. Ait-Ahmed O. Mech. Dev. 1997; 68: 91-100Crossref PubMed Scopus (33) Google Scholar). The probe was a PCR fragment between nucleotides 1637 and 2844 on the sequence of the I factor (GenBankTM accession number M14954) labeled with digoxigenin using the Nick translation kit from Roche Molecular Biochemicals. Protein detection by immunofluorescence on whole mount ovaries was performed using rabbit polyclonal antibodies against the C-terminal nine amino acids of the ORF1 protein of the I factor as described by Seleme et al. (28Seleme M. Busseau I. Malinsky S. Bucheton A. Teninges D. Genetics. 1999; 151: 761-771PubMed Google Scholar). We used the RNA circularization/RT-PCR technique (29Mandl C.W. Heinz F.X. Puchhammer-Stockl E. Kunz C. BioTechniques. 1991; 10: 484-486PubMed Google Scholar, 30Fromont-Racine M. Bertrand E. Pictet R. Grange T. Nucleic Acids Res. 1993; 21: 1683-1684Crossref PubMed Scopus (88) Google Scholar, 31Couttet P. Fromont-Racine M. Steel D. Pictet R. Grange T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5628-5633Crossref PubMed Scopus (187) Google Scholar) to analyze the transcripts produced by a fully active I factor. The I954 element (24Pritchard M.A. Dura J.M. Pelisson A. Bucheton A. Finnegan D.J. Mol Gen. Genet. 1988; 214: 533-540Crossref PubMed Scopus (40) Google Scholar) is an active I factor that was isolated from the wIR3 mutation (16Bucheton A. Paro R. Sang H.M. Pelisson A. Finnegan D.J. Cell. 1984; 38: 153-163Abstract Full Text PDF PubMed Scopus (188) Google Scholar). We introduced it into the Cha reactive strain. Because I factors transpose exclusively in the female germ line, transgenic lines were maintained by crossing transgenic males with virgin Cha females at each generation, thus ensuring that they contain a single copy of the I954 element. Total RNA was extracted from the dissected ovaries of females produced by crosses between transgenic males and Cha females, treated with tobacco acid pyrophosphatase to remove any CAP structure that would prevent ligation of the 5′-ends, and circularized as described under “Experimental Procedures.” Reverse transcription was primed using the oligonucleotide 2cir5′ (Fig. 1a), and two nested PCR reactions were performed, first using primers B290 and Ri160 and second using primers B95 and 2cir3′up (Fig. 1a). The products were analyzed on a 1.5% agarose gel (Fig. 1b). One major smeared band was obtained with RNA extracted from the ovaries of females issued from transgenic males along with some additional minor products of various sizes. None of these PCR products were obtained with RNA extracted from ovaries of Cha females, demonstrating that our conditions allow the specific detection of transcripts produced by the active I954 element. No amplification product was obtained when the tobacco acid pyrophosphatase step before RNA circularization was omitted (not shown), indicating that the transcripts produced by the I954 element are capped at the 5′-end like conventional mRNAs. To obtain information about the structure at the ends of the transcripts produced by the I954 element, we cloned and sequenced the major PCR products. The organization of the sequences of the RNA species are shown in Fig. 2. All start at the second nucleotide of I (Fig. 2a) and terminate with a poly(A) tail varying from 18 to 33 A residues (Fig. 2b). Two polyadenylation sites were used, which lie within the genomic DNA that is adjacent to the 3′-end of the element. We searched for consensus polyadenylation signals (AAUAAA) and G/U-rich sequences (32Graber J.H. Cantor C.R. Mohr S.C. Smith T.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14055-14060Crossref PubMed Scopus (213) Google Scholar, 33Chen F. MacDonald C.C. Wilusz J. Nucleic Acids Res. 1995; 23: 2614-2620Crossref PubMed Scopus (171) Google Scholar) in this region. There is no conventional AAUAAA sequence up to more than 50 nucleotides upstream of the two polyadenylation sites that were used. Possibly, the tandem UAA repeats could serve as a polyadenylation signal, and downstream G/U-rich sequences are present in the adjacent sequence (Fig. 2b). The 3′-ends of transcripts produced by the I954 element were also analyzed by conventional RT-PCR; reverse transcription was primed with an anchor oligo(dT) primer and followed by two sequential PCR amplifications using primers designed to amplify sequences between the 3′-end of I and the poly(A) stretch (see “Experimental Procedures”). The PCR products were cloned and sequenced. The results were similar to those obtained using the circularized RNAs; the five transcripts identified in this way are polyadenylated in the 3′-flanking sequences at one of the two positions determined previously by the RNA circularization/RT-PCR method (Fig. 2b). We designed I elements derived from I954 by addition of strong polyadenylation signals and/or removal of the tandem TAA repeats at the 3′-end (Fig. 2c). The ITK element is identical to I954 except in the 3′-flanking sequences, because the last TAA repeat is followed by a sequence containing the polyadenylation signals of the gene encoding the TK of the herpes simplex virus. The IΔ3TK element is identical to ITK except that the TAA repeats are deleted, leaving only one T residue. Finally, the IΔ3CC element derives from I954 by a deletion of the TAA repeats, leaving only one T plus two additional C residues that are fused to the EcoRI site of pUChsneo. Transgenic lines for these constructs were established and maintained in the same way as for I954. Total RNA was extracted from the dissected ovaries of females produced by crosses between transgenic males and Cha females. The 3′-ends of transcripts produced by the ITK, IΔ3TK, and IΔ3CC elements were analyzed by RT-PCR as described above for I954. The RT-PCR products were analyzed on a 1.5% agarose gel (Fig. 3). Several bands of low molecular weight (indicated by an asterisk in Fig. 3) were produced in all cases, including with RNA from the Cha reactive strain. These bands correspond to transcripts produced by the defective heterochromatic I elements that are present in all strains (15Bucheton A. Busseau I. Teninges D. Craig N.L. Craigie R. Gellert M. Lambowitz A. Mobile DNA II. ASM Press, Washington D. C.2002: 796-812Crossref Scopus (4) Google Scholar, 16Bucheton A. Paro R. Sang H.M. Pelisson A. Finnegan D.J. Cell. 1984; 38: 153-163Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar). Specific amplification products (indicated as A, B, and C in Fig. 3) were obtained after a RT-PCR of RNA from the ovaries of females issued from males transgenic for ITK, IΔ3TK, and IΔ3CC. These products were cloned and sequenced, and the data are shown in Fig. 2c. The ITK, IΔ3TK, and IΔ3CC elements produce transcripts of different classes according to the sites of polyadenylation. These different classes can be assigned to the different RT-PCR products observed on the gel (Fig. 3). Polyadenylation of transcripts produced by the ITK element occurs either within the flanking TK sequences or near the 3′-end of I (Fig. 2c), giving rise to the RT-PCR products designated A and B (in Fig. 3), respectively. Therefore, the presence of strong polyadenylation signals in the flanking sequences seem to efficiently determine the polyadenylation of a fraction of the transcripts. The IΔ3TK element produces the same classes of transcripts and, in addition, transcripts that are prematurely polyadenylated within the I element (Fig. 2c) corresponding to the RT-PCR products of slightly lower molecular weights (designated C, in Fig. 3). The IΔ3CC element produces transcripts that are polyadenylated in the 3′-flanking sequences and also transcripts that are prematurely polyadenylated within the I element (Fig. 2c), and give rise to the RT-PCR products designated B and C (in Fig. 3), respectively. We estimated the activity of the different I elements by determining their ability to induce female sterility. Females issued from crosses between Cha females and males transgenic for I954 or ITK were severely sterile. They produced no hatched progeny during the first 2 weeks of their adult lives and then became more and more fertile as they aged. These features are characteristics of the syndrome of sterility that is associated with high levels of retrotransposition of I factors (17Picard G. Mol. Gen. Genet. 1978; 164: 235-247Crossref Scopus (32) Google Scholar). This indicates that I954 and ITK are very active I factors that retrotranspose with high efficiency in the female germ line. In contrast, females issued from crosses between Cha females and males transgenic for IΔ3TK or IΔ3CC, deleted for the tandem TAA repeats, were normally fertile. Hatching percentages of the eggs were found to be 84 ± 9, 82 ± 10, and 86 ± 12 for three independent IΔ3TK transgenic lines and 86 ± 4, 81 ± 6, and 85 ± 8 for three independent IΔ3CC transgenic lines. This indicates that the IΔ3TK and IΔ3CC elements retrotranspose less than I954 and ITK. We verified that some retrotransposed copies of IΔ3TK and IΔ3CC could be detected by in situ hybridization experiments on salivary gland polytene chromosomes of larvae (data not shown). The transcripts and protein product of ORF1 synthesized by functional I elements co-localize in the female germ line and concentrate in the oocyte (15Bucheton A. Busseau I. Teninges D. Craig N.L. Craigie R. Gellert M. Lambowitz A. Mobile DNA II. ASM Press, Washington D. C.2002: 796-812Crossref Scopus (4) Google Scholar, 28Seleme M. Busseau I. Malinsky S. Bucheton A. Teninges D. Genetics. 1999; 151: 761-771PubMed Google Scholar). 2M.-C. Seleme, O. Disson, S. Chambeyron, S. Robin, C. Brun, I. Busseau, D. Teninges, and A. Bucheton, manuscript in preparation. The transcripts and ORF1 products of the I954 (Fig. 4, b and g) and ITK (Fig. 4, c and h) elements that retrotranspose with high efficiency show the expected pattern of expression. The same picture is also observed with the transcripts and ORF1 products of the IΔ3TK (Fig. 4, d and i) and IΔ3CC (Fig. 4,e and j) elements. This indicates that the transcripts produced by IΔ3TK and IΔ3CC are transported normally and translated. Therefore, IΔ3TK and IΔ3CC seem to be capable of building the components of the retrotransposition machinery. We recovered, by inverse PCR, transposed copies of the ITK, IΔ3TK, and IΔ3CC elements and determined the sequences at their 3′-ends (Fig. 2c). Transposed copies of ITK terminate with variable numbers of tandem TAA repeats. They do not contain sequences from the DNA flanking the progenitor element, indicating that reverse transcription starts within the UAA repeats of the RNA retrotransposition intermediate. Most transposed copies of IΔ3TK and IΔ3CC terminate a few nucleotides upstream or downstream of the 3′-end of the progenitor element. One of the copies produced by IΔ3TK ends within the 3′-UTR of I 104 base pairs upstream of the 3′-end and may have been generated by the reverse transcription of an RNA prematurely polyadenylated. Therefore, the tandem UAA repeats in the 3′-end of the retrotransposition intermediate of the I factor are necessary for the precision of the initiation of reverse transcription. Previous data (22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar, 25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar) suggested that a fraction of the transcripts produced by I factors may not be polyadenylated. The data (34De La Roche Saint Andre C. Bregliano J.C. Genetics. 1998; 148: 1875-1884Crossref PubMed Google Scholar) were obtained with studies of RNA extracted from whole flies, which might include some transcripts produced by I factors in somatic tissues and not correlated with retrotransposition. To focus our analysis on transcripts associated with I factor retrotransposition, we performed experiments on RNA extracted from dissected ovaries. We found that these transcripts undergo the classical maturation steps of messenger RNAs at the 5′- and 3′-ends; they are capped and polyadenylated. Transcription of the I factor is supposed to initiate at the first nucleotide. This expectation is supported by S1 mapping (22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar) and primer extension (21McLean C. Bucheton A. Finnegan D.J. Mol. Cell. Biol. 1993; 13: 1042-1050Crossref PubMed Scopus (85) Google Scholar) experiments, which mapped the transcription start site at the 5′-end of the I factor. However the resolution of these experiments did not allow us to determine precisely the exact first nucleotide of the transcript. All the RNA circularization/RT-PCR products that we have analyzed seem to derive from transcripts starting at the second nucleotide of the I954 element. This is puzzling because I954 produces complete retrotransposed copies, starting at the first deoxycytidine (24Pritchard M.A. Dura J.M. Pelisson A. Bucheton A. Finnegan D.J. Mol Gen. Genet. 1988; 214: 533-540Crossref PubMed Scopus (40) Google Scholar, 25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar). The first ribocytidine at the 5′-end of the transcripts might have been removed during the RNA circularization/RT-PCR procedure. Variable transcription start sites have been reported by other authors (35Kuhn J. Binder S. Nucleic Acids Res. 2002; 30: 439-446Crossref PubMed Scopus (73) Google Scholar, 36Sugahara Y. Carninci P. Itoh M. Shibata K. Konno H. Endo T. Muramatsu M. Hayashizaki Y. Gene (Amst.). 2001; 263: 93-102Crossref PubMed Scopus (43) Google Scholar) who used the RNA-circularization/RT-PCR or related techniques and can also be explained by removal of some nucleotides at the 5′-end of the transcripts during the procedures. Alternatively, it is not inconceivable that the transcription start site of the I factor is at the second nucleotide of the element and that an untemplated deoxycytidine would be added during reverse transcription or integration of the new copy by an unknown mechanism. We analyzed the polyadenylated transcripts of I elements with various flanking sequences at the 3′-end. Our results show that the polyadenylation sites are determined to some extent by cryptic consensus polyadenylation signals present in the flanking sequences. In some cases, the tandem UAA repeats transcribed from the I factor 3′-end appear to be used instead of the consensus AAUAAA polyadenylation signal. There is a AATAAA sequence within the 3′-UTR of the I factor 73 nucleotides upstream from the tandem TAA repeats (19Fawcett D.H. Lister C.K. Kellett E. Finnegan D.J. Cell. 1986; 47: 1007-1015Abstract Full Text PDF PubMed Scopus (190) Google Scholar). Noticeably, we found transcripts in which the polyadenylation site seems to be determined by this consensus signal in the case of the IΔ3TK and IΔ3CC elements that lack the tandem TAA repeats at the 3′-ends but not in the case of the I954 and ITK elements that have intact 3′-ends. This suggests that the tandem UAA repeats represent a stronger polyadenylation signal than the upstream AAUAAA consensus sequence. The latter is probably a very weak signal anyway because in the absence of tandem UAA repeats a substantial fraction of the transcripts undergo polyadenylation downstream of the 3′-end of the I element. The tandem TAA repeats at the 3′-end of the I factor are required for efficient and precise retrotransposition. The contrast between the ITK and the IΔ3TK elements that have identical 3′-flanking sequences and differ only by the presence or absence of tandem TAA repeats is particularly striking. Our results suggest that tandem UAA repeats in the transcript are important for its efficient utilization as a retrotransposition intermediate. The pattern of expression in the ovaries of transcripts that are devoid of tandem UAA repeats is not affected, and these transcripts are translated (Fig. 4). However, a moderate lowering in amounts would not have been detected by these experiments. Given that the tandem UAA repeats may be used as polyadenylation signals, it is possible that their deletion causes a drop in polyadenylation. The analysis of the rare transposed copies of IΔ3TK and IΔ3CC has been informative. Most of the copies terminate a few nucleotides upstream or downstream of the actual 3′-end of the progenitor element. One of these copies, resulting from retrotransposition of IΔ3TK, is truncated at the 3′-end at a position suggesting that it might result from the reverse transcription of an RNA polyadenylated within the 3′-UTR of the I element. In contrast, transposed copies generated by I elements ending with tandem TAA repeats always terminate precisely at the 3′-end of I with various numbers of tandem TAA repeats, as is usual for transposed copies of active I factors (24Pritchard M.A. Dura J.M. Pelisson A. Bucheton A. Finnegan D.J. Mol Gen. Genet. 1988; 214: 533-540Crossref PubMed Scopus (40) Google Scholar, 25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar, 37Busseau I. Malinsky S. Balakireva M. Chaboissier M.C. Teninges D. Bucheton A. Genetics. 1998; 148: 267-275Crossref PubMed Google Scholar). These results indicate that the tandem UAA repeats in the 3′-ends of the transcripts are required for the precise initiation of reverse transcription. This suggests that the reverse transcriptase of the I factor associates with the RNA transposition intermediate and recognizes the UAA repeats to initiate reverse transcription. In the absence of these repeats the reverse transcriptase would initiate inefficiently in a region of the RNA near the normal position of these repeats. These findings contrast with what is known about the initiation of reverse transcription in the case of human L1 elements (see below). The initiation of reverse transcription at the UAA repeats of the transcript implies that the sequences that are downstream of these repeats, including the poly(A) tail, are probably degraded during or after completion of the target-primed reverse transcription process. However, it cannot be excluded that these sequences are processed from the retrotransposition intermediate prior to reverse transcription either in the cytoplasm or in the nucleus. Noticeably, non-polyadenylated transcripts were identified in earlier studies (22Chaboissier M.C. Busseau I. Prosser J. Finnegan D.J. Bucheton A. EMBO J. 1990; 9: 3557-3563Crossref PubMed Scopus (71) Google Scholar,25Chaboissier M.C. Finnegan D. Bucheton A. Nucleic Acids Res. 2000; 28: 2467-2472Crossref PubMed Scopus (31) Google Scholar). It is noticeable that none of the transposed copies of the various I elements that we have studied in this paper contains a poly(dA) sequence at the 3′-end, indicating that the poly(A) tails of the transcripts cannot be used instead of the tandem UAA repeats for the initiation of reverse transcription. This contrasts with mammalian L1 elements for which reverse transcription is initiated in the poly(A) tails at the 3′-ends of the transcripts (12Moran J.V. DeBerardinis R.J. Kazazian H.H., Jr. Science. 1999; 283: 1530-1534Crossref PubMed Scopus (485) Google Scholar,13Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian H.H., Jr. Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar). The reverse transcription of L1 elements initiating in the poly(A) tail of read-through transcripts results in the insertion into new sites of sequences adjacent to the progenitor element along with the L1 sequences. Sequences resulting from this type of DNA transduction occur in the human and mouse genomes (38Pickeral O.K. Makalowski W. Boguski M.S. Boeke J.D. Genome Res. 2000; 10: 411-415Crossref PubMed Scopus (209) Google Scholar, 39Goodier J.L. Ostertag E.M. Kazazian H.H., Jr. Hum. Mol. Genet. 2000; 9: 653-657Crossref PubMed Scopus (196) Google Scholar). Therefore L1 elements are believed to represent a major source of shaping and evolution of mammalian genomes (40Kazazian H.H., Jr. Science. 2000; 289: 1152-1153Crossref PubMed Scopus (137) Google Scholar, 41Eickbush T. Science. 1999; 283: 1465-1467Crossref PubMed Google Scholar, 42Boeke J.D. Pickeral O.K. Nature. 1999; 398: 108-111Crossref PubMed Scopus (27) Google Scholar). In contrast, the reverse transcriptase of I factors initiates reverse transcription in the UAA repeats at the end of I element sequences. Consequently, the flanking sequences present in the transcripts are not inserted in the genome. Therefore, I elements are not expected to have the same influence as L1 elements in remodeling the genome. In addition, the weak specificity of the L1 retrotransposition machinery for L1 transcripts is thought to be responsible for the mobilization of short interspersed nucleotidic elements and the generation of processed pseudogenes (43Esnault C. Maestre J. Heidmann T. Nat. Genet. 2000; 24: 363-367Crossref PubMed Scopus (654) Google Scholar), which are important components of mammalian genomes. The retrotransposition machinery of the I factor seems to recognize strongly its own transcripts. It is therefore very unlikely that it is used for the reverse transcription of messenger RNAs or other transcripts. Strikingly, the Drosophila genome is devoid of short interspersed nucleotidic elements, and only very rare retropseudogenes have been identified in this organism (44Jeffs P. Ashburner M. Proc. R. Soc. Lond. B Biol. Sci. 1991; 244: 151-159Crossref PubMed Scopus (50) Google Scholar, 45Akhmanova A. Hennig W. Genome. 1998; 41: 396-401Crossref PubMed Scopus (4) Google Scholar). Therefore these variations in the retrotransposition mechanisms of non-LTR retrotransposons in mammals and Drosophila might account for major differences in the organization of the genomes of these species. The absence of short interspersed nucleotidic elements and processed pseudogenes in Drosophila might be due to the fact the I elements and other Drosophila retrotransposons are clean transposable elements. We thank Edouard Bertrand and François Rassendren for helpful advice during this work, Ounissa Ait-Ahmed and Martine Simonelig for discussions, and Martine Simonelig and Daniel Fisher for comments on the manuscript." @default.
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• W2071916965 title "Tandem UAA Repeats at the 3′-End of the Transcript Are Essential for the Precise Initiation of Reverse Transcription of the I Factor in Drosophila melanogaster" @default.
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