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J. Biol. Chem., Vol. 277, Issue 20, 17877-17882, May 17, 2002

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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^*

Séverine Chambeyron, Alain Bucheton, and Isabelle Busseau^§

From the Institut de Génétique Humaine, CNRS, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France

Received for publication, January 30, 2002, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Very little is known about the mechanism of retrotransposition of non-LTR¹ 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 (1). 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 (1-3). 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 (4-6) and maybe also in reverse transcription (7). In addition, their endonucleases have similarities with apurinic/apyrimidinic endonucleases (8, 9), whereas the endonuclease of R2 elements is related to bacterial restriction endonucleases (10). 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 (1, 11). 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 (12, 13). 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.

The I factor is a non-LTR retrotransposon of particular interest because its transposition occurs at high frequencies in "Inducer-Reactive" hybrid dysgenic females (14) (for a recent review see Ref. 15), 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 (14, 16). 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 (17).

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 (4). ORF2 encodes a putative polypeptide with endonuclease, reverse transcriptase, and RNase H domains (8, 18-20). I factors terminate at the 3'-ends by several (3-8) 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 (21). It produces a full-length 5.4-kb transcript, the abundance of which correlates with the transposition frequency (22). 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 (22, 23).

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Constructs and Transgenic Lines-- The pI954 construct has the I954 element inserted into the transformation vector pUChsneo (24). To generate ITK and I3TK, 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 I3CC, 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 (25).

Measurements of Female Sterility-- 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.

RNA Extraction-- 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.

RNA Circularization, Reverse Transcription, and PCR-- 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 M ammonium 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^TM 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.

Reverse Transcription with an Anchor-oligo(dT) Primer and PCR-- 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^TM 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.

Determination of Sequences at the 3'-Ends of Transposed Copies-- Genomic DNA was extracted from pools of 25 males using standard procedures. Inverse PCR experiments were performed as described by Chaboissier et al. (25), 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^TM Amp cloning kit and sequenced by Genome Express.

In Situ Hybridization and Immunofluorescence on Whole Mount Ovaries-- RNA detection by in situ hybridization on whole mount ovaries was performed following the protocol of Tautz and Pfeifle (26) adapted by Capri et al. (27). The probe was a PCR fragment between nucleotides 1637 and 2844 on the sequence of the I factor (GenBank^TM 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. (28).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcripts of the I Factor Are Capped and Polyadenylated-- We used the RNA circularization/RT-PCR technique (29-31) to analyze the transcripts produced by a fully active I factor. The I954 element (24) is an active I factor that was isolated from the w^IR3 mutation (16). 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.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Analyses of the transcripts of the I954 element using RNA circularization/RT-PCR experiments. a, positions of primers in the 5'- (left) and 3'-ends (right) of the I954 element. Thin white boxes represent untranslated regions, and wide white boxes represent coding regions. Oligonucleotide primers used in this work are indicated below as black arrowheads pointing in the direction of the DNA strand on which they map. b, an analysis on 1.5% agarose gels of the RNA circularization/RT-PCR products obtained using RNA extracted from ovaries of Cha females and ovaries of females issued from crosses between Cha females and males transgenic for I954 was performed. M, molecular markers (1-kb ladder from Invitrogen). Sizes are in base pairs (pb).

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 (32, 33) 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).

View larger version (75K): [in this window] [in a new window]   Fig. 2.   I elements, transcripts, and transposed copies. a, sequences at the 5'-ends of the I954 element and transcripts (RNAc, RNA sequence derived from four clones obtained using the RNA circularization/RT-PCR method) are shown. b, sequences at the 3'-ends of the I954 element and transcripts are shown (RNAc, same as above; RNA, RNA sequences derived from clones obtained using the anchor-oligo(dT) RT-PCR method). c, sequences at the 3'-ends of the ITK, I3TK, and I3CC elements, transcripts (RNA), and transposed copies (Copy) are shown. DNA sequences are in uppercase letters, and RNA sequences are in lowercase letters. Sequences shaded in gray correspond to sequences from the I factor in I954, ITK, I3TK, and I3CC and to sequences that are identical to those from the progenitor element in the transposed copies. Numbers separated by slashes at the 3'-ends of the RNA sequences indicate the numbers of adenyl residues found in independent clones. The consensus polyadenylation signals AATAAA are highlighted in black, and G/T-rich sequences are underlined on the DNA sequences of the I954, ITK, I3TK, and I3CC elements and flanking sequences.

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).

Sequences at the 3'-End of I, Including Tandem UAA Repeats, Influence the Site of Polyadenylation-- 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 I3TK element is identical to ITK except that the TAA repeats are deleted, leaving only one T residue. Finally, the I3CC 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, I3TK, and I3CC 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 (15, 16, 22). 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, I3TK, and I3CC. These products were cloned and sequenced, and the data are shown in Fig. 2c. The ITK, I3TK, and I3CC 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 I3TK 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 I3CC 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.

View larger version (96K): [in this window] [in a new window]   Fig. 3.   Analyses of the 3'-ends of transcripts using anchor-oligo(dT) RT-PCR experiments. An analysis on a 1.5% agarose gel of the RT-PCR products obtained using RNA extracted from ovaries of Cha females and females produced by crossing Cha females with males transgenic for ITK, I3TK and I3CC was performed. RT indicates a control experiment in which the reverse transcriptase step was omitted. M, molecular markers (1-kb ladder from Invitrogen). Sizes are in base pairs (pb). Arrows on the right indicate the presumed positions of products deriving from polyadenylation within the TK sequences adjacent to the 3'-ends of ITK and I3TK (A), products deriving from polyadenylation at the 3'-end of I (B), and products deriving from prematurely polyadenylated transcripts of ITK and I3CC (C). Asterisks indicate products deriving from transcripts of heterochromatic-defective I elements also present in RNAs extracted from ovaries of the Cha reactive strain.

The Tandem TAA Repeats at the 3'-End of I Are Required for Efficient Retrotransposition-- 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 (17). 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 I3TK or I3CC, 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 I3TK transgenic lines and 86 ± 4, 81 ± 6, and 85 ± 8 for three independent I3CC transgenic lines. This indicates that the I3TK and I3CC elements retrotranspose less than I954 and ITK. We verified that some retrotransposed copies of I3TK and I3CC 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 (15, 28).² 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 I3TK (Fig. 4, d and i) and I3CC (Fig. 4, e and j) elements. This indicates that the transcripts produced by I3TK and I3CC are transported normally and translated. Therefore, I3TK and I3CC seem to be capable of building the components of the retrotransposition machinery.

View larger version (58K): [in this window] [in a new window]   Fig. 4.   Expression of I elements in the ovaries. Left side, in situ hybridization experiments were performed on whole mount ovaries of Cha females (a) and females produced by crosses between Cha females and males transgenic for the I954 (b), ITK (c), I3TK (d), and I3CC (e) elements, using a digoxigenin-labeled PCR probe corresponding to an internal part of the I factor. Right side, fluorescent immunostaining was performed on whole mount ovaries of Cha females (f) and females produced by crosses between Cha females and males transgenic for the I954 (g), ITK (h), I3TK (i), and I3CC (j) elements, using anti-ORF1-C-terminal-nonapeptide antibodies.

The Tandem UAA Repeats in the Transcripts of I Are Required for the Precise Initiation of Reverse Transcription-- We recovered, by inverse PCR, transposed copies of the ITK, I3TK, and I3CC 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 I3TK and I3CC terminate a few nucleotides upstream or downstream of the 3'-end of the progenitor element. One of the copies produced by I3TK 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcripts of the I Factor Are Subject to the Classical Messenger RNA Maturation-- Previous data (22, 25) suggested that a fraction of the transcripts produced by I factors may not be polyadenylated. The data (34) 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 (22) and primer extension (21) 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 (24, 25). 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 (35, 36) 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 (19). Noticeably, we found transcripts in which the polyadenylation site seems to be determined by this consensus signal in the case of the I3TK and I3CC 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 UAA Repeats Determine the Site of Initiation of Reverse Transcription-- 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 I3TK 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 I3TK and I3CC 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 I3TK, 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 (24, 25, 37). 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 (22, 25).

Implications of the Properties of the Reverse Transcriptase of the I Factor in Genome Plasticity-- 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 (12, 13). 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 (38, 39). Therefore L1 elements are believed to represent a major source of shaping and evolution of mammalian genomes (40-42). 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 (43), 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 (44, 45). 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of fellowships from the Fondation pour la Recherche Médicale (FRM) and the Ministère de la Recherche et de la Technologie.

^§ To whom correspondence should be addressed. Tel.: 4-99-61-99-48; Fax: 4-99-61-99-01; E-mail: busseau@igh.cnrs.fr.

Published, JBC Papers in Press, March 6, 2002, DOI 10.1074/jbc.M200996200

² M.-C. Seleme, O. Disson, S. Chambeyron, S. Robin, C. Brun, I. Busseau, D. Teninges, and A. Bucheton, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: non-LTR, non-long terminal repeat; ORF, open reading frame; UTR, untranslated region; RT, reverse transcription; TK, thymidine kinase; Cha, Charolles reactive strain.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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