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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 knockdown increases the steady-state levels of Tbca13 RNA in GC-2spd(ts)-spermatocyte mouse cell line.After 48 h of transfection, total RNA was extracted from GC-2spd(ts) cells expressing non-target shRNA, TbcashRNA(knockdownTbca13+Tbca16) or Tbca16shRNA (to knockdown exclusively the Tbca16 RNA). Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca13 and Tbca16 mRNAs in Tbca shRNA expressing cells in comparison to those in cells expressing non-target shRNA. However, in cells expressing Tbca16 shRNA the steady-state levels of Tbca16 mRNA decrease whereas those of Tbca13 mRNA increase. Normalized cDNA is expressed as a percentage of maximal value (100%). *p<0,05 compared with control. The graphic bars are the mean±s.d. (error bars) of three independent assays. Values were standardized with those of Hprt cDNA. Statistical significance was calculated using a t-test.
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pone-0042536-g006: Tbca16 knockdown increases the steady-state levels of Tbca13 RNA in GC-2spd(ts)-spermatocyte mouse cell line.After 48 h of transfection, total RNA was extracted from GC-2spd(ts) cells expressing non-target shRNA, TbcashRNA(knockdownTbca13+Tbca16) or Tbca16shRNA (to knockdown exclusively the Tbca16 RNA). Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca13 and Tbca16 mRNAs in Tbca shRNA expressing cells in comparison to those in cells expressing non-target shRNA. However, in cells expressing Tbca16 shRNA the steady-state levels of Tbca16 mRNA decrease whereas those of Tbca13 mRNA increase. Normalized cDNA is expressed as a percentage of maximal value (100%). *p<0,05 compared with control. The graphic bars are the mean±s.d. (error bars) of three independent assays. Values were standardized with those of Hprt cDNA. Statistical significance was calculated using a t-test.

Mentions: Taking into consideration the expression patterns of the two Tbca genes during testis maturation we decided to study the effects of the specific Tbca16 knockdown in the GC-2spd(ts)-spermatocyte mouse cell line (GC-2 cells). As a control, GC-2 cells were transfected with a plasmid encoding a non-target shRNA to check for general non-specific effects associated with shRNA delivery and to confirm the sequence specificity of the silencing effect (negative control). Additionally, GC-2 cells were transfected with the Tbca shRNA coding plasmid to target the transcripts of both Tbca genes (13+16) and with a plasmid coding for the Tbca16 shRNA specifically targeting the transcripts of the Tbca16 gene. At 48 h post-transfection total RNAs were extracted from GC-2 cells expressing the non-target shRNA, the Tbca(13+16) shRNA, or the Tbca16shRNA which were then analyzed by RT-PCR. Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca16 mRNA in Tbca(13+16) and in Tbca16shRNA expressing cells, in comparison with those found in control cells expressing the non-target shRNA (Fig. 6). Also, a decrease in the Tbca13 mRNA steady-state levels was observed in cells transfected with the Tbca(13+16) shRNA. Remarkably, in cells expressing the Tbca16 shRNA we observed an increase in Tbca13 mRNA steady-state levels to levels higher than those found in cells expressing the non-target shRNA. The difference observed in Tbca16 mRNA decrease levels in cells transfected with Tbca(13+16) shRNA and Tbca16 shRNA could be explained by different silencing efficiencies of both interfering RNAs. Also, when GC-2 cells are transfected with a recombinant plasmid (see Material and Methods) to over-express the antiTbca16 transcript we observed an increase in the antiTbca16 transcript levels. The observed increase is accompanied by a decrease of about 20% on the Tbca13 transcript levels (Fig. S1).


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 knockdown increases the steady-state levels of Tbca13 RNA in GC-2spd(ts)-spermatocyte mouse cell line.After 48 h of transfection, total RNA was extracted from GC-2spd(ts) cells expressing non-target shRNA, TbcashRNA(knockdownTbca13+Tbca16) or Tbca16shRNA (to knockdown exclusively the Tbca16 RNA). Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca13 and Tbca16 mRNAs in Tbca shRNA expressing cells in comparison to those in cells expressing non-target shRNA. However, in cells expressing Tbca16 shRNA the steady-state levels of Tbca16 mRNA decrease whereas those of Tbca13 mRNA increase. Normalized cDNA is expressed as a percentage of maximal value (100%). *p<0,05 compared with control. The graphic bars are the mean±s.d. (error bars) of three independent assays. Values were standardized with those of Hprt cDNA. Statistical significance was calculated using a t-test.
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Related In: Results  -  Collection

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pone-0042536-g006: Tbca16 knockdown increases the steady-state levels of Tbca13 RNA in GC-2spd(ts)-spermatocyte mouse cell line.After 48 h of transfection, total RNA was extracted from GC-2spd(ts) cells expressing non-target shRNA, TbcashRNA(knockdownTbca13+Tbca16) or Tbca16shRNA (to knockdown exclusively the Tbca16 RNA). Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca13 and Tbca16 mRNAs in Tbca shRNA expressing cells in comparison to those in cells expressing non-target shRNA. However, in cells expressing Tbca16 shRNA the steady-state levels of Tbca16 mRNA decrease whereas those of Tbca13 mRNA increase. Normalized cDNA is expressed as a percentage of maximal value (100%). *p<0,05 compared with control. The graphic bars are the mean±s.d. (error bars) of three independent assays. Values were standardized with those of Hprt cDNA. Statistical significance was calculated using a t-test.
Mentions: Taking into consideration the expression patterns of the two Tbca genes during testis maturation we decided to study the effects of the specific Tbca16 knockdown in the GC-2spd(ts)-spermatocyte mouse cell line (GC-2 cells). As a control, GC-2 cells were transfected with a plasmid encoding a non-target shRNA to check for general non-specific effects associated with shRNA delivery and to confirm the sequence specificity of the silencing effect (negative control). Additionally, GC-2 cells were transfected with the Tbca shRNA coding plasmid to target the transcripts of both Tbca genes (13+16) and with a plasmid coding for the Tbca16 shRNA specifically targeting the transcripts of the Tbca16 gene. At 48 h post-transfection total RNAs were extracted from GC-2 cells expressing the non-target shRNA, the Tbca(13+16) shRNA, or the Tbca16shRNA which were then analyzed by RT-PCR. Semi-quantitative RT-PCR analysis showed a decrease in the steady-state levels of Tbca16 mRNA in Tbca(13+16) and in Tbca16shRNA expressing cells, in comparison with those found in control cells expressing the non-target shRNA (Fig. 6). Also, a decrease in the Tbca13 mRNA steady-state levels was observed in cells transfected with the Tbca(13+16) shRNA. Remarkably, in cells expressing the Tbca16 shRNA we observed an increase in Tbca13 mRNA steady-state levels to levels higher than those found in cells expressing the non-target shRNA. The difference observed in Tbca16 mRNA decrease levels in cells transfected with Tbca(13+16) shRNA and Tbca16 shRNA could be explained by different silencing efficiencies of both interfering RNAs. Also, when GC-2 cells are transfected with a recombinant plasmid (see Material and Methods) to over-express the antiTbca16 transcript we observed an increase in the antiTbca16 transcript levels. The observed increase is accompanied by a decrease of about 20% on the Tbca13 transcript levels (Fig. S1).

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