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Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons.

Maxwell PH, Belote JM, Levis RW - Nucleic Acids Res. (2006)

Bottom Line: These results are consistent with the production of an array of TART transcripts.A sequence overlapping this sense start site contains a good match to an initiator consensus for the transcription start sites of Drosophila LTR retrotransposons.Our results highlight differences between transcription of TART and of other Drosophila non-LTR elements and they provide a foundation for testing the relationship between exceptional aspects of TART transcription and TART's specialized role at telomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244, USA. pmaxwell@wadsworth.org

ABSTRACT
The Drosophila non-long terminal repeat (non-LTR) retrotransposons TART and HeT-A specifically retrotranspose to chromosome ends to maintain Drosophila telomeric DNA. Relatively little is known, though, about the regulation of their expression and their retrotransposition to telomeres. We have used rapid amplification of cDNA ends (RACE) to identify multiple transcription initiation and polyadenylation sites for sense and antisense transcripts of three subfamilies of TART elements in Drosophila melanogaster. These results are consistent with the production of an array of TART transcripts. In contrast to other Drosophila non-LTR elements, a major initiation site for sense transcripts was mapped near the 3' end of the TART 5'-untranslated region (5'-UTR), rather than at the start of the 5'-UTR. A sequence overlapping this sense start site contains a good match to an initiator consensus for the transcription start sites of Drosophila LTR retrotransposons. Interestingly, analysis of 5' RACE products for antisense transcripts and the GenBank EST database revealed that TART antisense transcripts contain multiple introns. Our results highlight differences between transcription of TART and of other Drosophila non-LTR elements and they provide a foundation for testing the relationship between exceptional aspects of TART transcription and TART's specialized role at telomeres.

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5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites (18). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.
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fig3: 5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites (18). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.

Mentions: We initially expected to map a TART 5′ end within the first few nucleotides of TART sequences or in previously unidentified upstream 5′-UTR sequences, so we took steps to confirm the reliability of the 5′ RACE mapping. The results of RT–PCR experiments using different combinations of primers upstream and downstream of the site of 5a were consistent with most TART transcripts initiating at site 5a, rather than at sites further upstream in the 5′-UTR (data not shown). In addition, we used our samples and RACE method to map HeT-A transcription initiation sites. HeT-A transcription start sites were previously mapped to HeT-A 3′-UTR sequences from a second element upstream of the element being transcribed, at positions −62 and −31 [(18), the C and A denoted with circles in Figure 3]. We mapped HeT-A 5′ ends to positions −93 and −31 in the 3′-UTR using adult RNA (Figure 3, lines with squares) and to position −61 using larval RNA (Figure 3, line with a circle). The −93 site we mapped could be a site that is used in flies but not in S2 cells (the −62 and −31 sites were mapped in S2 cells) or it could be that the RACE method we used was able to map a longer 5′ end than the primer extension method used previously (18). Our ability to confirm the transcription start sites of HeT-A suggests that our 5′ RACE mapping of TART was reliable.


Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons.

Maxwell PH, Belote JM, Levis RW - Nucleic Acids Res. (2006)

5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites (18). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636488&req=5

fig3: 5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites (18). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.
Mentions: We initially expected to map a TART 5′ end within the first few nucleotides of TART sequences or in previously unidentified upstream 5′-UTR sequences, so we took steps to confirm the reliability of the 5′ RACE mapping. The results of RT–PCR experiments using different combinations of primers upstream and downstream of the site of 5a were consistent with most TART transcripts initiating at site 5a, rather than at sites further upstream in the 5′-UTR (data not shown). In addition, we used our samples and RACE method to map HeT-A transcription initiation sites. HeT-A transcription start sites were previously mapped to HeT-A 3′-UTR sequences from a second element upstream of the element being transcribed, at positions −62 and −31 [(18), the C and A denoted with circles in Figure 3]. We mapped HeT-A 5′ ends to positions −93 and −31 in the 3′-UTR using adult RNA (Figure 3, lines with squares) and to position −61 using larval RNA (Figure 3, line with a circle). The −93 site we mapped could be a site that is used in flies but not in S2 cells (the −62 and −31 sites were mapped in S2 cells) or it could be that the RACE method we used was able to map a longer 5′ end than the primer extension method used previously (18). Our ability to confirm the transcription start sites of HeT-A suggests that our 5′ RACE mapping of TART was reliable.

Bottom Line: These results are consistent with the production of an array of TART transcripts.A sequence overlapping this sense start site contains a good match to an initiator consensus for the transcription start sites of Drosophila LTR retrotransposons.Our results highlight differences between transcription of TART and of other Drosophila non-LTR elements and they provide a foundation for testing the relationship between exceptional aspects of TART transcription and TART's specialized role at telomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244, USA. pmaxwell@wadsworth.org

ABSTRACT
The Drosophila non-long terminal repeat (non-LTR) retrotransposons TART and HeT-A specifically retrotranspose to chromosome ends to maintain Drosophila telomeric DNA. Relatively little is known, though, about the regulation of their expression and their retrotransposition to telomeres. We have used rapid amplification of cDNA ends (RACE) to identify multiple transcription initiation and polyadenylation sites for sense and antisense transcripts of three subfamilies of TART elements in Drosophila melanogaster. These results are consistent with the production of an array of TART transcripts. In contrast to other Drosophila non-LTR elements, a major initiation site for sense transcripts was mapped near the 3' end of the TART 5'-untranslated region (5'-UTR), rather than at the start of the 5'-UTR. A sequence overlapping this sense start site contains a good match to an initiator consensus for the transcription start sites of Drosophila LTR retrotransposons. Interestingly, analysis of 5' RACE products for antisense transcripts and the GenBank EST database revealed that TART antisense transcripts contain multiple introns. Our results highlight differences between transcription of TART and of other Drosophila non-LTR elements and they provide a foundation for testing the relationship between exceptional aspects of TART transcription and TART's specialized role at telomeres.

Show MeSH