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

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.

Figure 1. 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 program (C). The aminoacid differences are indicated in the table, accordingly to the aminoacid position.

Figure 2. Study of Tbca16 and Tbca13expression in different mouse tissues by RT-PCR.

RT-PCR analysis of Tbca13 and Tbca16 transcript levels using total RNA samples extracted from different mouse tissues (3 mice, 25 post-natal days old, were used in this analysis). In contrast with Tbca13 expression, Tbca16 is not highly expressed in testis. The Hprt expression was analyzed as an endogenous control.

Figure 3. 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.

Figure 4. 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).

Figure 5. 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.

Figure 6. 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.