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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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Related in: MedlinePlus

TBCA16 protein is absent in early stages of mouse spermatogenesis.Protein extracts from mouse testis in early stages of spermatogenesis (14 post-natal days – PND14) and recombinant TBCA13 and TBCA16 proteins produced and purified from bacteria were analyzed by 16.5% (w/v) Tricine–SDS–PAGE followed by western blot with a specific polyclonal antibody directed to TBCA. Note that this antibody recognizes both recombinant proteins. Under these conditions, TBCA13 and TBCA16 proteins have distinct motilities. The approximate molecular mass of the proteins is indicated at the left side of the panels. The regions around the 14 kDa marked by dashed squares were excised and proteins present in these regions were analyzed by electrospray mass spectrometry (MS/MS). The analyses lead to the identification of the TBCA13 specific peptide R.LEAAYTDLQQILESEK.D (the specific aminoacid is underlined). No TBCA16 specific peptides were identified (see table).
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pone-0042536-g004: TBCA16 protein is absent in early stages of mouse spermatogenesis.Protein extracts from mouse testis in early stages of spermatogenesis (14 post-natal days – PND14) and recombinant TBCA13 and TBCA16 proteins produced and purified from bacteria were analyzed by 16.5% (w/v) Tricine–SDS–PAGE followed by western blot with a specific polyclonal antibody directed to TBCA. Note that this antibody recognizes both recombinant proteins. Under these conditions, TBCA13 and TBCA16 proteins have distinct motilities. The approximate molecular mass of the proteins is indicated at the left side of the panels. The regions around the 14 kDa marked by dashed squares were excised and proteins present in these regions were analyzed by electrospray mass spectrometry (MS/MS). The analyses lead to the identification of the TBCA13 specific peptide R.LEAAYTDLQQILESEK.D (the specific aminoacid is underlined). No TBCA16 specific peptides were identified (see table).

Mentions: The fact that both Tbca13 and Tbca16 genes present distinct expression patterns during testis development led us to investigate if the steady-state levels of the respective encoded proteins accompanied the levels of the specific transcripts. In fact, the production of an mRNA encoded by the Tbca16 gene did not imply by itself the existence of a TBCA16 protein. Given the fact that the TBCA13 and TBCA16 predicted proteins differ only in 4 amino-acid residues it would be difficult to produce a specific antibody capable of distinguishing them. To overcome this difficulty and to identify the TBCA16 protein in vivo we cloned the coding regions of Tbca13 and Tbca16 in a bacterial expression vector and expressed them in E. coli. The recombinant proteins were purified and analyzed by electrophoresis on a Tricine-SDS-PAGE in parallel with a 14 post-natal days testis protein extract. This testis developmental stage was chosen since it corresponds to the stage with the highest Tbca16 mRNA levels (Fig. 3). This analysis was followed by western blot using an antibody against human TBCA that recognized the mouse TBCA13 and TBCA16 proteins produced in bacteria (Fig. 4). Interestingly, although both proteins present the same predicted molecular mass, under the conditions used, TBCA16 migrated faster than TBCA13 (Fig. 4). Thus, it was possible to distinguish the two proteins due to their distinct mobility in Tricine-SDS-PAGE. However, in 14 post-natal days testis protein extracts the antibody only recognized a unique protein band presenting a migration behavior similar to that of TBCA13 (Fig. 4). This suggested that, although these protein extracts corresponded to the testis maturation stage were Tbca16 is up-regulated, they only appeared to contain the protein product of the Tbca13 gene. To confirm this result we first re-analyzed protein extracts from 14 days old testis in parallel with purified TBCA13 and TBCA16 proteins in a Tricine-SDS-PAGE followed by Coomassie blue staining. In this analysis the purified TBCA13 and 16 proteins were used as migration references to allow the identification of the region where both TBCA proteins should be present in the analyzed testis protein extracts. Subsequently, this region was excised from the gel and the proteins presented there were identified by tandem mass spectrometry. To make sure that this analysis was able to distinguish between the two TBCA proteins, purified TBCA13 and 16 proteins were previously analyzed. The obtained data revealed that the distinction between TBCA13 and TBCA16 proteins by tandem mass spectrometry was possible due to the aminoacid substitutions occurring in position 77 and 88. In fact, the theoretical complete trypsin hydrolysis of the two TBCA proteins originated two distinct peptides with different molecular masses (TBCA13:LEAAYTDLQQILESEK and TBCA16:QLEAAYTGLQQILESEK). Since the identification of the proteins analyzed by tandem mass spectrometry requires a search in protein databases to identify the specific protein profile we have updated them by introducing the corresponding TBCA16 data. Noteworthy, with this approach we were only able to detect the specific peptide corresponding to the TBCA13 protein in protein extracts of 14 days post-natal mouse testis (Fig. 4). The LEAAYTDLQQILESEK peptide was identified with a peptide score of 80, clearly above the identity threshold (51) determined by the search algorithm, thus confirming the unambiguous identification of TBCA13 in the sample. Although the peptide QLEAAYTGLQQILESEK was not detected, the possibility of minimum amounts of TBCA16 being synthesized cannot be completely excluded. Furthermore this result is in agreement with the ones obtained by western blot (Fig. 4) indicating that the TBCA16 protein is most probably not being synthesized in this testis maturation stage.


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 protein is absent in early stages of mouse spermatogenesis.Protein extracts from mouse testis in early stages of spermatogenesis (14 post-natal days – PND14) and recombinant TBCA13 and TBCA16 proteins produced and purified from bacteria were analyzed by 16.5% (w/v) Tricine–SDS–PAGE followed by western blot with a specific polyclonal antibody directed to TBCA. Note that this antibody recognizes both recombinant proteins. Under these conditions, TBCA13 and TBCA16 proteins have distinct motilities. The approximate molecular mass of the proteins is indicated at the left side of the panels. The regions around the 14 kDa marked by dashed squares were excised and proteins present in these regions were analyzed by electrospray mass spectrometry (MS/MS). The analyses lead to the identification of the TBCA13 specific peptide R.LEAAYTDLQQILESEK.D (the specific aminoacid is underlined). No TBCA16 specific peptides were identified (see table).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412815&req=5

pone-0042536-g004: TBCA16 protein is absent in early stages of mouse spermatogenesis.Protein extracts from mouse testis in early stages of spermatogenesis (14 post-natal days – PND14) and recombinant TBCA13 and TBCA16 proteins produced and purified from bacteria were analyzed by 16.5% (w/v) Tricine–SDS–PAGE followed by western blot with a specific polyclonal antibody directed to TBCA. Note that this antibody recognizes both recombinant proteins. Under these conditions, TBCA13 and TBCA16 proteins have distinct motilities. The approximate molecular mass of the proteins is indicated at the left side of the panels. The regions around the 14 kDa marked by dashed squares were excised and proteins present in these regions were analyzed by electrospray mass spectrometry (MS/MS). The analyses lead to the identification of the TBCA13 specific peptide R.LEAAYTDLQQILESEK.D (the specific aminoacid is underlined). No TBCA16 specific peptides were identified (see table).
Mentions: The fact that both Tbca13 and Tbca16 genes present distinct expression patterns during testis development led us to investigate if the steady-state levels of the respective encoded proteins accompanied the levels of the specific transcripts. In fact, the production of an mRNA encoded by the Tbca16 gene did not imply by itself the existence of a TBCA16 protein. Given the fact that the TBCA13 and TBCA16 predicted proteins differ only in 4 amino-acid residues it would be difficult to produce a specific antibody capable of distinguishing them. To overcome this difficulty and to identify the TBCA16 protein in vivo we cloned the coding regions of Tbca13 and Tbca16 in a bacterial expression vector and expressed them in E. coli. The recombinant proteins were purified and analyzed by electrophoresis on a Tricine-SDS-PAGE in parallel with a 14 post-natal days testis protein extract. This testis developmental stage was chosen since it corresponds to the stage with the highest Tbca16 mRNA levels (Fig. 3). This analysis was followed by western blot using an antibody against human TBCA that recognized the mouse TBCA13 and TBCA16 proteins produced in bacteria (Fig. 4). Interestingly, although both proteins present the same predicted molecular mass, under the conditions used, TBCA16 migrated faster than TBCA13 (Fig. 4). Thus, it was possible to distinguish the two proteins due to their distinct mobility in Tricine-SDS-PAGE. However, in 14 post-natal days testis protein extracts the antibody only recognized a unique protein band presenting a migration behavior similar to that of TBCA13 (Fig. 4). This suggested that, although these protein extracts corresponded to the testis maturation stage were Tbca16 is up-regulated, they only appeared to contain the protein product of the Tbca13 gene. To confirm this result we first re-analyzed protein extracts from 14 days old testis in parallel with purified TBCA13 and TBCA16 proteins in a Tricine-SDS-PAGE followed by Coomassie blue staining. In this analysis the purified TBCA13 and 16 proteins were used as migration references to allow the identification of the region where both TBCA proteins should be present in the analyzed testis protein extracts. Subsequently, this region was excised from the gel and the proteins presented there were identified by tandem mass spectrometry. To make sure that this analysis was able to distinguish between the two TBCA proteins, purified TBCA13 and 16 proteins were previously analyzed. The obtained data revealed that the distinction between TBCA13 and TBCA16 proteins by tandem mass spectrometry was possible due to the aminoacid substitutions occurring in position 77 and 88. In fact, the theoretical complete trypsin hydrolysis of the two TBCA proteins originated two distinct peptides with different molecular masses (TBCA13:LEAAYTDLQQILESEK and TBCA16:QLEAAYTGLQQILESEK). Since the identification of the proteins analyzed by tandem mass spectrometry requires a search in protein databases to identify the specific protein profile we have updated them by introducing the corresponding TBCA16 data. Noteworthy, with this approach we were only able to detect the specific peptide corresponding to the TBCA13 protein in protein extracts of 14 days post-natal mouse testis (Fig. 4). The LEAAYTDLQQILESEK peptide was identified with a peptide score of 80, clearly above the identity threshold (51) determined by the search algorithm, thus confirming the unambiguous identification of TBCA13 in the sample. Although the peptide QLEAAYTGLQQILESEK was not detected, the possibility of minimum amounts of TBCA16 being synthesized cannot be completely excluded. Furthermore this result is in agreement with the ones obtained by western blot (Fig. 4) indicating that the TBCA16 protein is most probably not being synthesized in this testis maturation stage.

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