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The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation.

Nolasco S, Bellido J, Gonçalves J, Tavares A, Zabala JC, Soares H - PLoS ONE (2012)

Bottom Line: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16).These puzzling results led us to re-analyze the expression of Tbca16.We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Medicina, IFIMAV-Universidad de Cantabria, Santander, Spain.

ABSTRACT

Background: Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules.

Methodology/principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line.

Conclusions/significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

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Tbca16 gene codes for a sense and for an antisense transcript.Total RNA was extracted from 14 post-natal days mouse testis and analyzed by RT-PCR. For the cDNA synthesis oligodT (cDNA-oligodT), Tbca16 sequence-specific primer for antisense orientation (cDNA-antisense) or Tbca16 sequence-specific primer for sense orientation (cDNA-sense) were used. The presence of the Tbca16 transcript was analyzed by PCR using specific primers. In every case a single band with the expected size was detected. A PCR performed only with RNA (RNA) as the template was done as a control to detect any residual amplification from a DNA genomic contamination.
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pone-0042536-g005: Tbca16 gene codes for a sense and for an antisense transcript.Total RNA was extracted from 14 post-natal days mouse testis and analyzed by RT-PCR. For the cDNA synthesis oligodT (cDNA-oligodT), Tbca16 sequence-specific primer for antisense orientation (cDNA-antisense) or Tbca16 sequence-specific primer for sense orientation (cDNA-sense) were used. The presence of the Tbca16 transcript was analyzed by PCR using specific primers. In every case a single band with the expected size was detected. A PCR performed only with RNA (RNA) as the template was done as a control to detect any residual amplification from a DNA genomic contamination.

Mentions: These intriguing results associated with the observation that Tbca16 presents an opposite expression pattern to that of Tbca13 lead us to put forward the hypothesis that Tbca16 transcripts could play a regulatory role in the expression of the Tbca13 gene. Moreover, there are growing evidences in the literature showing that the expression of genes can be regulated by non-coding antisense mRNAs, some transcribed from their pseudogenes [29]. In our previous strategy to analyze the specific expression of Tbca13 and Tbca16 genes by RT-PCR (Fig. 2 and 3) the cDNAs were synthesized using a primer d(T) to hybridize with the poly(A)+ tail, which did not allow us to account for the orientation of the RNAs under study. To address this issue we set up an RT-PCR experiment in which the cDNA would be produced using a sequence-specific primer complementary to the Tbca16 mRNA in a sense (P-specific sense) and antisense orientation (P-specific antisense) (see Fig. 1A). Subsequently, we used DNase-treated RNAs from 14 post-natal days mouse testis to synthesize cDNA either using the specific sense- and antisense-Tbca16 primers, or the universal d(T) primer. Next, Tbca16 transcripts were amplified by PCR using the specific primers for Tbca16 (Fig. 1A, P-specific Tbca16) and using the following templates: (i) DNase treated RNA used in the cDNA synthesis; (ii) a cDNA synthesized using the d(T) primer and (iii) cDNAs synthesized with the specific Tbca16 primer in sense or antisense orientation. The results presented in Fig. 5 show that there is no amplification when the RNA sample is used as a template showing that the RNA sample was not contaminated with genomic DNA. On the other hand, for each cDNA sample used, a single band with the expected size for the Tbca16 transcript was detected (Fig. 5). These PCR products were sequenced and their nucleotide sequence was an exact match to the sequence of Tbca16 transcripts leading to the conclusion that the Tbca16 gene is transcribed both in a sense and an anti-sense (anti-Tbca16) orientation.


The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation.

Nolasco S, Bellido J, Gonçalves J, Tavares A, Zabala JC, Soares H - PLoS ONE (2012)

Tbca16 gene codes for a sense and for an antisense transcript.Total RNA was extracted from 14 post-natal days mouse testis and analyzed by RT-PCR. For the cDNA synthesis oligodT (cDNA-oligodT), Tbca16 sequence-specific primer for antisense orientation (cDNA-antisense) or Tbca16 sequence-specific primer for sense orientation (cDNA-sense) were used. The presence of the Tbca16 transcript was analyzed by PCR using specific primers. In every case a single band with the expected size was detected. A PCR performed only with RNA (RNA) as the template was done as a control to detect any residual amplification from a DNA genomic contamination.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412815&req=5

pone-0042536-g005: Tbca16 gene codes for a sense and for an antisense transcript.Total RNA was extracted from 14 post-natal days mouse testis and analyzed by RT-PCR. For the cDNA synthesis oligodT (cDNA-oligodT), Tbca16 sequence-specific primer for antisense orientation (cDNA-antisense) or Tbca16 sequence-specific primer for sense orientation (cDNA-sense) were used. The presence of the Tbca16 transcript was analyzed by PCR using specific primers. In every case a single band with the expected size was detected. A PCR performed only with RNA (RNA) as the template was done as a control to detect any residual amplification from a DNA genomic contamination.
Mentions: These intriguing results associated with the observation that Tbca16 presents an opposite expression pattern to that of Tbca13 lead us to put forward the hypothesis that Tbca16 transcripts could play a regulatory role in the expression of the Tbca13 gene. Moreover, there are growing evidences in the literature showing that the expression of genes can be regulated by non-coding antisense mRNAs, some transcribed from their pseudogenes [29]. In our previous strategy to analyze the specific expression of Tbca13 and Tbca16 genes by RT-PCR (Fig. 2 and 3) the cDNAs were synthesized using a primer d(T) to hybridize with the poly(A)+ tail, which did not allow us to account for the orientation of the RNAs under study. To address this issue we set up an RT-PCR experiment in which the cDNA would be produced using a sequence-specific primer complementary to the Tbca16 mRNA in a sense (P-specific sense) and antisense orientation (P-specific antisense) (see Fig. 1A). Subsequently, we used DNase-treated RNAs from 14 post-natal days mouse testis to synthesize cDNA either using the specific sense- and antisense-Tbca16 primers, or the universal d(T) primer. Next, Tbca16 transcripts were amplified by PCR using the specific primers for Tbca16 (Fig. 1A, P-specific Tbca16) and using the following templates: (i) DNase treated RNA used in the cDNA synthesis; (ii) a cDNA synthesized using the d(T) primer and (iii) cDNAs synthesized with the specific Tbca16 primer in sense or antisense orientation. The results presented in Fig. 5 show that there is no amplification when the RNA sample is used as a template showing that the RNA sample was not contaminated with genomic DNA. On the other hand, for each cDNA sample used, a single band with the expected size for the Tbca16 transcript was detected (Fig. 5). These PCR products were sequenced and their nucleotide sequence was an exact match to the sequence of Tbca16 transcripts leading to the conclusion that the Tbca16 gene is transcribed both in a sense and an anti-sense (anti-Tbca16) orientation.

Bottom Line: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16).These puzzling results led us to re-analyze the expression of Tbca16.We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Medicina, IFIMAV-Universidad de Cantabria, Santander, Spain.

ABSTRACT

Background: Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules.

Methodology/principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line.

Conclusions/significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

Show MeSH
Related in: MedlinePlus