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Transcription of the rat testis-specific Rtdpoz-T1 and -T2 retrogenes during embryo development: co-transcription and frequent exonisation of transposable element sequences.

Huang CJ, Lin WY, Chang CM, Choo KB - BMC Mol. Biol. (2009)

Bottom Line: The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR).In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence.Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, School of Agriculture, Chinese Culture University, Yang-Ming-Shan, Taipei, Taiwan. hqr2@faculty.pccu.edu.tw

ABSTRACT

Background: Retrotransposition is an important evolutionary force for the creation of new and potentially functional intronless genes which are collectively called retrogenes. Many retrogenes are expressed in the testis and the gene products have been shown to actively participate in spermatogenesis and other unique functions of the male germline. We have previously reported a cluster of retrogenes in the rat genome that encode putative TRAF- and POZ-domain proteins. Two of the genes, Rtdpoz-T1 and -T2 (abbreviated as T1 and T2), have further been shown to be expressed specifically in the rat testis.

Results: We show here that the T1 and T2 genes are also expressed in the rat embryo up to days 16-17 of development when the genes are silenced until being re-activated in the adult testis. On database interrogation, we find that some T1/T2 exons are chromosomally duplicated as cassettes of 2 or 3 exons consistent with retro-duplication. The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR). Most importantly, the T1/T2 locus is embedded within a dense field of relics of transposable element (TE) derived mainly from LINE1 and ERV sequences, and the TE sequences are frequently exonised through alternative splicing to form the 5'-UTR sequences of the T1/T2 transcripts. In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence. Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed. We further demonstrate that the exonised 5'-UTR TE sequences could lead to the creation of upstream open reading frames resulting in translational repression.

Conclusion: Exonisation of TE sequences is a frequent event in the transcription of retrogenes during embryonic development and in the testis and may contribute to post-transcriptional regulation of expression of retrogenes.

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Detection of T1–T2 chimeric transcripts in the testis. (A) RT-PCR profiling of T1–T2 chimeric transcripts. Two rounds of nested PCR were performed using oligo(dT)-primed RT products of the testis mRNA. In the first-round PCR, the consensus Ex1a-B and the T1- or T2-specific 3'-UTR primers, T1SP-R1 and T2SP-R1, respectively, were used (see Figure 1A for relative positions and Table 2 for sequence details); in the second-round PCR, the exon 3 (T1) or exon 2 (T2)-based T2SP-F1 or T1SP-F1 primers were used in combination with T1SP-R1 and T2SP-R1. The T1 and T2 plasmids were used as controls in both rounds of PCR (lanes 1–2 and 4–5). The PCR products (with designations in brackets) generated in the second-round PCR (lane 6) were cloned and sequenced. (B) Schematic depiction of the PCR products derived from (A). The exons of the transcripts are shown using the same colour code and exon designation as in Figure 2. Primers used in the second-round PCR are also shown.
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Figure 4: Detection of T1–T2 chimeric transcripts in the testis. (A) RT-PCR profiling of T1–T2 chimeric transcripts. Two rounds of nested PCR were performed using oligo(dT)-primed RT products of the testis mRNA. In the first-round PCR, the consensus Ex1a-B and the T1- or T2-specific 3'-UTR primers, T1SP-R1 and T2SP-R1, respectively, were used (see Figure 1A for relative positions and Table 2 for sequence details); in the second-round PCR, the exon 3 (T1) or exon 2 (T2)-based T2SP-F1 or T1SP-F1 primers were used in combination with T1SP-R1 and T2SP-R1. The T1 and T2 plasmids were used as controls in both rounds of PCR (lanes 1–2 and 4–5). The PCR products (with designations in brackets) generated in the second-round PCR (lane 6) were cloned and sequenced. (B) Schematic depiction of the PCR products derived from (A). The exons of the transcripts are shown using the same colour code and exon designation as in Figure 2. Primers used in the second-round PCR are also shown.

Mentions: In our previous work, we presented a testicular transcript which was a composite of T1 and T2 sequences [20]. To authenticate T1-T2 chimerism in this work, RT-PCR was performed using a T1- or T2-exon 2-specific forward primer in mix-gene combinations with a T2 or T1 reverse primers located at the 3'-UTR of the respective gene for detection of possible T1-T2 or T2-T1 exon constitutions. To increase sensitivity and specificity, two rounds of nested PCR were performed: the first-round PCR was done using an exon 1a primer (Ex1a-B in Figure 1A) common to both T1 and T2, and a T1 or T2 3'-UTR sequence-specific reverse primer (T1SP-R1 or T1SP-R2 in Figure 1A). In the PCR, plasmids carrying the T1 or T2 cDNA sequence were included as controls; these plasmids generated positive bands corresponding to the respective gene indicating gene specificity (Figure 4A, lanes 1 and 2). When the testicular mRNA was used in the first-round RT-PCR using the T1 or T2 primers, two major bands were discerned (Figure 4A, lane 3). For subsequent PCR, T1- or T2-specific exon 2 forward primer (T2SP-F1 or T1SP-F1) was used in a mix-gene fashion in combination with the T2- or T1-specific reverse primer (T1SP-R1 or T2SP-R2) located at the 3'-UTR sequence of the respective gene (Figure 4B; see also Figure 1A for primer map positions). In the control experiments in which the T1 and T2 plasmids were also tested in the mix-gene reactions, no PCR products were detected as expected for such mono-gene scenarios (Figure 4A, lanes 4 and 5). However, when the testicular first-round cDNA products were subjected to the mix-gene PCR, two distinct bands were now discerned in each of the mix-gene reactions (Figure 4A, lane 6).


Transcription of the rat testis-specific Rtdpoz-T1 and -T2 retrogenes during embryo development: co-transcription and frequent exonisation of transposable element sequences.

Huang CJ, Lin WY, Chang CM, Choo KB - BMC Mol. Biol. (2009)

Detection of T1–T2 chimeric transcripts in the testis. (A) RT-PCR profiling of T1–T2 chimeric transcripts. Two rounds of nested PCR were performed using oligo(dT)-primed RT products of the testis mRNA. In the first-round PCR, the consensus Ex1a-B and the T1- or T2-specific 3'-UTR primers, T1SP-R1 and T2SP-R1, respectively, were used (see Figure 1A for relative positions and Table 2 for sequence details); in the second-round PCR, the exon 3 (T1) or exon 2 (T2)-based T2SP-F1 or T1SP-F1 primers were used in combination with T1SP-R1 and T2SP-R1. The T1 and T2 plasmids were used as controls in both rounds of PCR (lanes 1–2 and 4–5). The PCR products (with designations in brackets) generated in the second-round PCR (lane 6) were cloned and sequenced. (B) Schematic depiction of the PCR products derived from (A). The exons of the transcripts are shown using the same colour code and exon designation as in Figure 2. Primers used in the second-round PCR are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Detection of T1–T2 chimeric transcripts in the testis. (A) RT-PCR profiling of T1–T2 chimeric transcripts. Two rounds of nested PCR were performed using oligo(dT)-primed RT products of the testis mRNA. In the first-round PCR, the consensus Ex1a-B and the T1- or T2-specific 3'-UTR primers, T1SP-R1 and T2SP-R1, respectively, were used (see Figure 1A for relative positions and Table 2 for sequence details); in the second-round PCR, the exon 3 (T1) or exon 2 (T2)-based T2SP-F1 or T1SP-F1 primers were used in combination with T1SP-R1 and T2SP-R1. The T1 and T2 plasmids were used as controls in both rounds of PCR (lanes 1–2 and 4–5). The PCR products (with designations in brackets) generated in the second-round PCR (lane 6) were cloned and sequenced. (B) Schematic depiction of the PCR products derived from (A). The exons of the transcripts are shown using the same colour code and exon designation as in Figure 2. Primers used in the second-round PCR are also shown.
Mentions: In our previous work, we presented a testicular transcript which was a composite of T1 and T2 sequences [20]. To authenticate T1-T2 chimerism in this work, RT-PCR was performed using a T1- or T2-exon 2-specific forward primer in mix-gene combinations with a T2 or T1 reverse primers located at the 3'-UTR of the respective gene for detection of possible T1-T2 or T2-T1 exon constitutions. To increase sensitivity and specificity, two rounds of nested PCR were performed: the first-round PCR was done using an exon 1a primer (Ex1a-B in Figure 1A) common to both T1 and T2, and a T1 or T2 3'-UTR sequence-specific reverse primer (T1SP-R1 or T1SP-R2 in Figure 1A). In the PCR, plasmids carrying the T1 or T2 cDNA sequence were included as controls; these plasmids generated positive bands corresponding to the respective gene indicating gene specificity (Figure 4A, lanes 1 and 2). When the testicular mRNA was used in the first-round RT-PCR using the T1 or T2 primers, two major bands were discerned (Figure 4A, lane 3). For subsequent PCR, T1- or T2-specific exon 2 forward primer (T2SP-F1 or T1SP-F1) was used in a mix-gene fashion in combination with the T2- or T1-specific reverse primer (T1SP-R1 or T2SP-R2) located at the 3'-UTR sequence of the respective gene (Figure 4B; see also Figure 1A for primer map positions). In the control experiments in which the T1 and T2 plasmids were also tested in the mix-gene reactions, no PCR products were detected as expected for such mono-gene scenarios (Figure 4A, lanes 4 and 5). However, when the testicular first-round cDNA products were subjected to the mix-gene PCR, two distinct bands were now discerned in each of the mix-gene reactions (Figure 4A, lane 6).

Bottom Line: The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR).In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence.Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, School of Agriculture, Chinese Culture University, Yang-Ming-Shan, Taipei, Taiwan. hqr2@faculty.pccu.edu.tw

ABSTRACT

Background: Retrotransposition is an important evolutionary force for the creation of new and potentially functional intronless genes which are collectively called retrogenes. Many retrogenes are expressed in the testis and the gene products have been shown to actively participate in spermatogenesis and other unique functions of the male germline. We have previously reported a cluster of retrogenes in the rat genome that encode putative TRAF- and POZ-domain proteins. Two of the genes, Rtdpoz-T1 and -T2 (abbreviated as T1 and T2), have further been shown to be expressed specifically in the rat testis.

Results: We show here that the T1 and T2 genes are also expressed in the rat embryo up to days 16-17 of development when the genes are silenced until being re-activated in the adult testis. On database interrogation, we find that some T1/T2 exons are chromosomally duplicated as cassettes of 2 or 3 exons consistent with retro-duplication. The embryonic T1/T2 transcripts, characterised by RT-PCR-cloning and rapid amplification of cDNA ends, are further found to have acquired one or more noncoding exons in the 5'-untranslated region (5'-UTR). Most importantly, the T1/T2 locus is embedded within a dense field of relics of transposable element (TE) derived mainly from LINE1 and ERV sequences, and the TE sequences are frequently exonised through alternative splicing to form the 5'-UTR sequences of the T1/T2 transcripts. In a case of T1 transcript, the 3'-end is extended into and terminated within an L1 sequence. Since the two genes share a common exon 1 and are, therefore, regulated by a single promoter, a T2-to-T1 co-transcription model is proposed. We further demonstrate that the exonised 5'-UTR TE sequences could lead to the creation of upstream open reading frames resulting in translational repression.

Conclusion: Exonisation of TE sequences is a frequent event in the transcription of retrogenes during embryonic development and in the testis and may contribute to post-transcriptional regulation of expression of retrogenes.

Show MeSH
Related in: MedlinePlus