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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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Study of Tbca16 and Tbca13 expression pattern during mouse spermatogenesis.Total RNA was obtained from mouse testis at different stages of maturation (3 sets of 14, 18 and 25 post-natal days old mice were used in this study). The Tbca16 and Tbca13 expression levels were analyzed by semi-quantitative RT-PCR and normalized by the expression of Hprt. During spermatogenesis the steady-state levels of Tbca13 mRNA increase whereas a decrease in the steady-state levels of Tbca16 mRNA is observed. Normalized cDNA levels are expressed as a percentage of maximal value (100%) for the 14 post-natal day. The p values were determined in comparison to the 14 post-natal days. The graphic bars are the mean±s.d. (error bars) of three independent assays. Statistical significance was calculated using a t-test.
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pone-0042536-g003: Study of Tbca16 and Tbca13 expression pattern during mouse spermatogenesis.Total RNA was obtained from mouse testis at different stages of maturation (3 sets of 14, 18 and 25 post-natal days old mice were used in this study). The Tbca16 and Tbca13 expression levels were analyzed by semi-quantitative RT-PCR and normalized by the expression of Hprt. During spermatogenesis the steady-state levels of Tbca13 mRNA increase whereas a decrease in the steady-state levels of Tbca16 mRNA is observed. Normalized cDNA levels are expressed as a percentage of maximal value (100%) for the 14 post-natal day. The p values were determined in comparison to the 14 post-natal days. The graphic bars are the mean±s.d. (error bars) of three independent assays. Statistical significance was calculated using a t-test.

Mentions: Previous studies, where no distinction between the two specific Tbca transcripts was made, have shown that Tbca was more abundantly expressed in testis than other adult tissues. In fact, Tbca was progressively up-regulated from the onset of meiosis throughout spermatogenesis, being more abundant in differentiating spermatids [44]. This Tbca expression pattern was proposed to be associated to microtubule cytoskeleton changes and β-tubulin processing throughout spermatogenesis rather than to meiosis [44]. The existence of two mouse genes encoding two distinct TBCA isotypes prompted us to investigate if both genes were transcribed and presented tissue-specific regulation. Therefore, we extracted total RNA from different mouse organs and performed RT-PCR using specific forward primers for each Tbca gene. Each specific primer was designed to hybridize with a sequence located about 66 bp upstream of the start codon at 5′-UTR (5′-untranslated region see Fig. 1A, P-specific Tbca16) of the two Tbca genes, where these sequences started to diverge. Before any analysis, a search of each of these specific primers throughout the mice genome sequence was performed. This BLAST analysis showed these primers to be specific for the 5′-UTR of each Tbca gene. The reverse primer was the same for both genes and hybridizes with a conserved sequence localized close to the stop codon (see Fig. 1A. P-Tbca reverse). The obtained amplification products were sequenced and this analysis showed that these products correspond to the amplification of the expected transcripts from the gene under analysis. To exclude the possibility of genomic DNA contamination affecting these results, all RNA samples were treated with DNase I and tested by PCR using primers specific for genomic DNA, prior to cDNA synthesis. The obtained results showed that Tbca13, as well as Tbca16 are both transcribed at different levels in all the mouse organs analyzed (Fig. 2). This analysis also revealed that, contrary to Tbca16, Tbca13 is highly expressed in adult testis in comparison with the other organs. This shows that the high TBCA levels previously observed in mature testis [44] correspond to Tbca13. Given what was previously described we decided to analyze also the expression of Tbca13 and Tbca16 genes during the different stages of the mouse first spermatogenesis, which occurs during the first post-natal month in parallel with testis maturation. Consequently, we extracted total RNAs from mouse testis at different post-natal days (14, 18 and 25 days) and analyzed the expression of both Tbca genes by RT-PCR using the primers already described (Fig. 3). With this analysis we observed that Tbca13 and Tbca16 genes present opposite expression patterns during the process of testis maturation. In fact, Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. Taken together, these data indicate that the two genes are regulated differentially and suggest that the products encoded by them might play distinct roles during testis development.


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)

Study of Tbca16 and Tbca13 expression pattern during mouse spermatogenesis.Total RNA was obtained from mouse testis at different stages of maturation (3 sets of 14, 18 and 25 post-natal days old mice were used in this study). The Tbca16 and Tbca13 expression levels were analyzed by semi-quantitative RT-PCR and normalized by the expression of Hprt. During spermatogenesis the steady-state levels of Tbca13 mRNA increase whereas a decrease in the steady-state levels of Tbca16 mRNA is observed. Normalized cDNA levels are expressed as a percentage of maximal value (100%) for the 14 post-natal day. The p values were determined in comparison to the 14 post-natal days. The graphic bars are the mean±s.d. (error bars) of three independent assays. Statistical significance was calculated using a t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412815&req=5

pone-0042536-g003: Study of Tbca16 and Tbca13 expression pattern during mouse spermatogenesis.Total RNA was obtained from mouse testis at different stages of maturation (3 sets of 14, 18 and 25 post-natal days old mice were used in this study). The Tbca16 and Tbca13 expression levels were analyzed by semi-quantitative RT-PCR and normalized by the expression of Hprt. During spermatogenesis the steady-state levels of Tbca13 mRNA increase whereas a decrease in the steady-state levels of Tbca16 mRNA is observed. Normalized cDNA levels are expressed as a percentage of maximal value (100%) for the 14 post-natal day. The p values were determined in comparison to the 14 post-natal days. The graphic bars are the mean±s.d. (error bars) of three independent assays. Statistical significance was calculated using a t-test.
Mentions: Previous studies, where no distinction between the two specific Tbca transcripts was made, have shown that Tbca was more abundantly expressed in testis than other adult tissues. In fact, Tbca was progressively up-regulated from the onset of meiosis throughout spermatogenesis, being more abundant in differentiating spermatids [44]. This Tbca expression pattern was proposed to be associated to microtubule cytoskeleton changes and β-tubulin processing throughout spermatogenesis rather than to meiosis [44]. The existence of two mouse genes encoding two distinct TBCA isotypes prompted us to investigate if both genes were transcribed and presented tissue-specific regulation. Therefore, we extracted total RNA from different mouse organs and performed RT-PCR using specific forward primers for each Tbca gene. Each specific primer was designed to hybridize with a sequence located about 66 bp upstream of the start codon at 5′-UTR (5′-untranslated region see Fig. 1A, P-specific Tbca16) of the two Tbca genes, where these sequences started to diverge. Before any analysis, a search of each of these specific primers throughout the mice genome sequence was performed. This BLAST analysis showed these primers to be specific for the 5′-UTR of each Tbca gene. The reverse primer was the same for both genes and hybridizes with a conserved sequence localized close to the stop codon (see Fig. 1A. P-Tbca reverse). The obtained amplification products were sequenced and this analysis showed that these products correspond to the amplification of the expected transcripts from the gene under analysis. To exclude the possibility of genomic DNA contamination affecting these results, all RNA samples were treated with DNase I and tested by PCR using primers specific for genomic DNA, prior to cDNA synthesis. The obtained results showed that Tbca13, as well as Tbca16 are both transcribed at different levels in all the mouse organs analyzed (Fig. 2). This analysis also revealed that, contrary to Tbca16, Tbca13 is highly expressed in adult testis in comparison with the other organs. This shows that the high TBCA levels previously observed in mature testis [44] correspond to Tbca13. Given what was previously described we decided to analyze also the expression of Tbca13 and Tbca16 genes during the different stages of the mouse first spermatogenesis, which occurs during the first post-natal month in parallel with testis maturation. Consequently, we extracted total RNAs from mouse testis at different post-natal days (14, 18 and 25 days) and analyzed the expression of both Tbca genes by RT-PCR using the primers already described (Fig. 3). With this analysis we observed that Tbca13 and Tbca16 genes present opposite expression patterns during the process of testis maturation. In fact, Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. Taken together, these data indicate that the two genes are regulated differentially and suggest that the products encoded by them might play distinct roles during testis development.

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