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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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Comparison of Tbca13 and Tbca16 sequences and 3D Model Structure of TBCA16 and TBCA13.Comparison between nucleotide sequences of Tbca16 and Tbca13 (A). The alignment shows 7 differences inside the coding region (the different nucleotides are in red, start and stop codons are inside a red dashed box). The regions where the different primers/shRNAs were designed are indicated. (B) Comparison of the aminoacid sequences of the putative TBCA16 and theTBCA13 protein, aminoacids are colored according to their polarity. The 4 differences are signaled with a black dot above the respective aminoacid (B). The sequences in (A) and (B) were done using the CLC Sequence Viewer 6.5.3 Program. (C) TBCA13 and TBCA16 3D models obtained using the program PyMOL [55] program (C). The aminoacid differences are indicated in the table, accordingly to the aminoacid position.
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pone-0042536-g001: Comparison of Tbca13 and Tbca16 sequences and 3D Model Structure of TBCA16 and TBCA13.Comparison between nucleotide sequences of Tbca16 and Tbca13 (A). The alignment shows 7 differences inside the coding region (the different nucleotides are in red, start and stop codons are inside a red dashed box). The regions where the different primers/shRNAs were designed are indicated. (B) Comparison of the aminoacid sequences of the putative TBCA16 and theTBCA13 protein, aminoacids are colored according to their polarity. The 4 differences are signaled with a black dot above the respective aminoacid (B). The sequences in (A) and (B) were done using the CLC Sequence Viewer 6.5.3 Program. (C) TBCA13 and TBCA16 3D models obtained using the program PyMOL [55] program (C). The aminoacid differences are indicated in the table, accordingly to the aminoacid position.

Mentions: TBCA is a β-tubulin binding protein that participates in the tubulin folding pathway. In vitro, TBCA is not critical for tubulin folding but enhances β-tubulin dimerization [41]. On the other hand, our previous studies of TBCA loss-of-function showed that human TBCA is an essential gene in human cell lines, its depletion causing a decrease in α- and β-tubulin levels [43]. These data indicate that TBCA is important for microtubule formation and consequently for microtubule-dependent functions. This is also hinted by the fact that TBCA is highly expressed in testis and is up-regulated during spermatogenesis, a process in which the microtubule cytoskeleton is preponderant. These observations lead us to go further in the functional characterization of Tbca in vivo. In the course of these studies we have detected that the mouse genome possess two Tbca genes, one previously described localized in chromosome 13 (Tbca13) and an uncharacterized Tbca gene in chromosome 16 (Tbca16). Tbca16 is localized inside the intron 3–4 of the Adenylatecyclase 9 gene (Adcy9) being its putative coding sequence in the same strand as the Adcy9 exons. The nucleotide sequence analysis of the two Tbca genes revealed that their structure is different. The Tbca13 gene presents its coding region interrupted by three introns. In contrast the Tbca16 gene is intronless. A comparison between the nucleotide sequences of both genes revealed that their coding sequences present a high degree of identity (98%) with only 7 nucleotide substitutions (Fig. 1A). This high degree of nucleotide sequence identity extends to the 5′and 3′-noncoding regions. For example, upstream of the ATG codon, the two genes present indistinguishable nucleotide sequences over 30 nucleotides and present an identity of 97% over 174 nucleotides of the 3′-non-coding region (Fig. 1A). As a consequence, the two Tbca genes encode two putative closely related proteins differing only in 4 aminoacid residues (Fig. 1B). These substitutions may not have significant impact in the 3D structure of the putative TBCA16 protein, given the similarity of their predicted 3D structures (Fig. 1C). Even so, important charged residues for putative protein interactions would disappear in the hypothetical TBCA16 protein.


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)

Comparison of Tbca13 and Tbca16 sequences and 3D Model Structure of TBCA16 and TBCA13.Comparison between nucleotide sequences of Tbca16 and Tbca13 (A). The alignment shows 7 differences inside the coding region (the different nucleotides are in red, start and stop codons are inside a red dashed box). The regions where the different primers/shRNAs were designed are indicated. (B) Comparison of the aminoacid sequences of the putative TBCA16 and theTBCA13 protein, aminoacids are colored according to their polarity. The 4 differences are signaled with a black dot above the respective aminoacid (B). The sequences in (A) and (B) were done using the CLC Sequence Viewer 6.5.3 Program. (C) TBCA13 and TBCA16 3D models obtained using the program PyMOL [55] program (C). The aminoacid differences are indicated in the table, accordingly to the aminoacid position.
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pone-0042536-g001: Comparison of Tbca13 and Tbca16 sequences and 3D Model Structure of TBCA16 and TBCA13.Comparison between nucleotide sequences of Tbca16 and Tbca13 (A). The alignment shows 7 differences inside the coding region (the different nucleotides are in red, start and stop codons are inside a red dashed box). The regions where the different primers/shRNAs were designed are indicated. (B) Comparison of the aminoacid sequences of the putative TBCA16 and theTBCA13 protein, aminoacids are colored according to their polarity. The 4 differences are signaled with a black dot above the respective aminoacid (B). The sequences in (A) and (B) were done using the CLC Sequence Viewer 6.5.3 Program. (C) TBCA13 and TBCA16 3D models obtained using the program PyMOL [55] program (C). The aminoacid differences are indicated in the table, accordingly to the aminoacid position.
Mentions: TBCA is a β-tubulin binding protein that participates in the tubulin folding pathway. In vitro, TBCA is not critical for tubulin folding but enhances β-tubulin dimerization [41]. On the other hand, our previous studies of TBCA loss-of-function showed that human TBCA is an essential gene in human cell lines, its depletion causing a decrease in α- and β-tubulin levels [43]. These data indicate that TBCA is important for microtubule formation and consequently for microtubule-dependent functions. This is also hinted by the fact that TBCA is highly expressed in testis and is up-regulated during spermatogenesis, a process in which the microtubule cytoskeleton is preponderant. These observations lead us to go further in the functional characterization of Tbca in vivo. In the course of these studies we have detected that the mouse genome possess two Tbca genes, one previously described localized in chromosome 13 (Tbca13) and an uncharacterized Tbca gene in chromosome 16 (Tbca16). Tbca16 is localized inside the intron 3–4 of the Adenylatecyclase 9 gene (Adcy9) being its putative coding sequence in the same strand as the Adcy9 exons. The nucleotide sequence analysis of the two Tbca genes revealed that their structure is different. The Tbca13 gene presents its coding region interrupted by three introns. In contrast the Tbca16 gene is intronless. A comparison between the nucleotide sequences of both genes revealed that their coding sequences present a high degree of identity (98%) with only 7 nucleotide substitutions (Fig. 1A). This high degree of nucleotide sequence identity extends to the 5′and 3′-noncoding regions. For example, upstream of the ATG codon, the two genes present indistinguishable nucleotide sequences over 30 nucleotides and present an identity of 97% over 174 nucleotides of the 3′-non-coding region (Fig. 1A). As a consequence, the two Tbca genes encode two putative closely related proteins differing only in 4 aminoacid residues (Fig. 1B). These substitutions may not have significant impact in the 3D structure of the putative TBCA16 protein, given the similarity of their predicted 3D structures (Fig. 1C). Even so, important charged residues for putative protein interactions would disappear in the hypothetical TBCA16 protein.

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