Communicated by Victor A. McKusick, Johns Hopkins University School of Medicine, Baltimore, MD, December 22, 2006 (received for review June 16, 2006)
The human TXNDC3 gene and related products. (A) TXNDC3 cDNA structure showing the location of the exons drawn to scale. The translation start and stop codons are labeled with ATG and TAA, respectively. The translated region is hashed. Exon 7 is underlined in blue, and intron 6 is shown as a thin line below exons 6 and 7. The red asterisks mark the locations of the c.271–27C>T and c.1277T>A nucleotide variations, located in intron 6 and exon 15, respectively. (B) Structure of the TXNDC3 isoforms. The TXNDC3fl isoform (Upper), and the TXNDC3d7 isoform (Lower). The thioredoxin (TRX) domain and the two NDK domains are shown in yellow and green, respectively. Within the TRX domain, the active site (GCPC) is shown by an orange box, and, within the NDK domains, the putative NDP kinase active sites are shown by pink boxes. The location of the region encoded by exon 7 is underlined in blue. The location of the p.Leu426X mutation is shown by a red asterisk.
Electron micrograph of cross-sections of respiratory cilia. (Left) Normal ciliary ultrastructure. (Right) Two cross-sections of respiratory cilia from patient D50X1 showing a heterogeneous ultrastructure: whereas some cilia appear normal (Left), others (66%) display outer dynein arm defects (Right). The black arrow indicates a normal outer dynein arm, and white arrowheads indicate shorter or missing outer dynein arms. (Scale bars: 100 nm.)
In vivo expression of TXNDC3fl and TXNDC3d7 isoforms. (A) Expression of the TXNDC3fl and TXNDC3d7 isoforms by means of conventional RT-PCR using primers F1 and R1 located in exons 6 and 8, respectively. RT-PCR amplifications were performed on RNAs extracted from human testis, trachea, and nasal cells, and from lymphoblastoid cell lines established for all members of family D50 (D50F, D50M, D50X1, D50S1, and D50S2), for patient D65X1 with a c.271–27C/T genotype, and for 15 controls, C1 to C15, with C1 to C14 having a c.271–27C/C genotype and C15 having a c.271–27C/T genotype. One representative experiment of three independent experiments is shown. L, 1-kb+ ladder; Neg, reaction without RNA. (B) Relative expression of TXNDC3fl and TXNDC3d7 transcripts generated by conventional PCR. The amounts of TXNDC3fl and TXNDC3d7 transcripts, calculated by the GeneTools program, are represented with black and gray bars, respectively. The sum of the two isoforms was arbitrarily set to 1. (Left) The results for individuals who carry the c.271–27C/C genotype, i.e., the patient's mother (D50M) and brother (D50S2) and controls (C1 to C14). (Right) The results for the five individuals bearing the c.271–27C>T variant, i.e., patient D50X1, her father (D50F), her brother (D50S1), patient D65X1, and control C15. Results are represented as means ± SEM of three independent experiments.
Impact of the c.271–27C>T variant on splicing of TXNDC3 transcripts. (A) Schematic representation of the different TXNDC3 constructs used in this experiment. Exons and introns are represented by open boxes and thin lines, respectively. The location of the c.271–27C/T SNP is indicated by a black arrowhead. The primers used in the RT-PCR experiments (F1 and R1) are represented by horizontal arrows. (B) RT-PCR amplification of TXNDC3 transcripts from HeLa cells transfected with minigenes carrying either a cytosine in intron 6 (i.e., pTXNDC3_C1, pTXNDC3_C2, pTXNDC3_T1>C, and pTXNDC3_T2>C) or a thymine at that position (i.e., pTXNDC3_T1, pTXNDC3_T2, pTXNDC3_C1>T, and pTXNDC3_C2>T) (see Materials and Methods). L, 1-kb+ ladder.
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Duriez et al. 10.1073/pnas.0611405104. |
Fig. 5. Identification of TXNDC3 mutations in family D50 and their intrafamilial segregation. The blackened symbol indicates the patient (D50X1), and empty symbols indicate unaffected family members (D50M, D50F, D50S1, and D50S2). Under each individual are shown the electrophoregrams of the two regions spanning the mutated sites (Int.6 and Ex.15 stand for intron 6 and exon 15). The c.1277T>A mutation identified in exon 15 (black arrowhead) would replace a leucine residue at codon 426 by a stop codon (p.Leu426X). The c.271-27C>T variant located within intron 6 is shown by a white arrowhead.
Fig. 6. PCR quantification of in vivo expression of TXNDC3fl and TXNDC3d7 isoforms. (A) Results of the PCR quantification by Genetools program enable calculation of the R ratios (TXNDC3fl/[TXNDC3fl + TXNDC3d7]) that are expressed as mean values of three different experiments. (B) Expression of TXNDC3fl and TXNDC3d7 transcripts in family D50 and in patient D65X1, as well as in control samples, as assessed by means of real-time quantitative RT-PCR. The relative ratio of TXNDC3fl transcripts vs. TXNDC3d7 transcripts was calculated with the formula r' = 2-DD Ct (see SI Materials and Methods). Values (R') are the mean ± SEM of three to four independent experiments performed in triplicate.
Fig. 7. (A) Evolutionary tree of the TXNDC3 proteins. For each species, the overall amino acid identity shared with the human sequence is shown. (B) Sequence alignment of the residues encoded by the human TXNDC3 exon 7(TXNDC3 E7) and the TXNDC6 exon 5 (TXNDC6 E5). Residues that are identical and similar are highlighted in dark and light blue, respectively. (C) Multiple sequence alignment of the residues encoded by the human TXNDC3 exon 7 and the corresponding exons from Ciona intestinalis to the chimpanzee. Residues that are identical and similar to the human sequence are highlighted in dark and light blue, respectively.
Fig. 8. Comparison of the binding of TXNDC3fl and TXNDC3d7 to microtubules. In vitro translated TXNDC3fl and TXNDC3d7 isoforms were incubated in the presence of polymerized tubulin. The percentage of specifically bound TXNDC3d7 (bound TXNDC3d7/[bound TXNDC3d7 + free TXNDC3d7]) is presented as a function of the percentage of specifically bound TXNDC3fl (bound TXNDC3fl/[bound TXNDC3fl + free TXNDC3fl]). For each independent experiment plotted here (open circles), the binding of each isoform was determined by means of a spin down assay (see SI Materials and Methods). The data obtained fit with a linear regression function (y = ax + b, with a = 1.33, b = 1.76 and a standard deviation of 1.7).
SI Materials and Methods
Real-Time Quantitative RT-PCR. Real-time quantitative RT-PCR was performed on an ABI PRISM 7000 Sequence Detector with the SYBR Green PCR Master mix (Applied Biosystems, Weiterstadt, Germany). Two primer sets were used: one (F2 and R2) detecting only the TXNDC3fl isoform by annealing in the region deleted in the alternatively spliced variant and the other (F3 and R3) detecting both the TXNDC3fl and TXNDC3d7 isoforms. Expression of TXNDC3 isoforms compared to b2 microglobulin RNA was evaluated with the comparative threshold method (Ct). Each experiment was done in triplicate; triplicate values were averaged, and averaged Ct values for b2 microglobulin were subtracted from each value to give the DCt. For each sample, the total amount of TXNDC3fl and TXNDC3d7 transcripts was chosen as the calibrator that was subtracted from the DCt of the TXNDC3fl isoform to give DDCt. The relative amount of the TXNDC3fl isoform compared to the calibrator was calculated by the formula r' = 2-DDCt. For each sample, the r' values of three to four independent experiments were averaged, to determine a ratio (R') of the TXNDC3fl isoform to the calibrator expressed as mean ±SEM.
Constructs. Total RNA from human testis was used as template to amplify by means of RT-PCR the TXNDC3fl sequence with primers F7 and R7. The resulting cDNA was cloned into the pCRII-TOPO vector (Invitrogen, Paisley, United Kingdom), generating pCR_TXNDC3fl that was subsequently subjected to site-directed mutagenesis with primers F8 and R8 to create a plasmid containing the TXNDC3d7 cDNA sequence (pCR_TXNDC3d7). The XhoI/KpnI digest of these two latter plasmids generated the TXNDC3fl and TXNDC3d7 cDNAs that were subsequently cloned into the pTNT vector (Promega, Mannheim, Germany) to generate pTNT_TXNDC3fl and pTNT_TXNDC3d7 convenient for in vitro transcription-translation experiments.
In Vitro Transcription and Translation and in Vitro Microtubule-Binding Assays. pTNT_TXNDC3fl and pTNT_TXNDC3d7 were used as templates to produce the corresponding recombinant proteins through the use of the TNT Quick Coupled in vitro Transcription/Translation Systems kit (Promega, Mannheim, Germany) in the presence of [35S]methionine and [35S]cysteine. In vitro microtubule-binding assays were performed with pure polymerized microtubules made from purified tubulin (Cytoskeleton, Denver, CO) in the presence of radiolabeled recombinant TXNDC3fl and TXNDC3d7 proteins, according to the manufacturer's instructions. Briefly, each TXNDC3 isoform was incubated with polymerized microtubules at 25°C for 30 min in the presence of 2 mM AMP-PNP, 0.5 mM GTP and 22 mM Taxol. The mixture was centrifuged at 47,000 g for 60 min at 20°C. Supernatants were saved. Two subsequent pelleting reactions were performed. Aliquots of both supernatants and final pellets were resolved on SDS-PAGE and analyzed by use of a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA). The ImageQuant software was used for quantification. As controls, the same experiments were performed in the absence of microtubules.
Phylogenetic Tree and Sequence Alignments. The protein sequences used for phylogenetic analysis are human TXNDC3 and orthologs from chimpanzee, dog, cow, mouse, rat, chicken, frog, pufferfish, tetraodon, sea urchin and ciona (SI Table 2). Before multiple alignments, we corrected all of these sequences, except the human and dog sequences. Actually, most of them were partial or exhibited discrepancy between Ensembl and NCBI versions, or included amino acid residues highly divergent from all remaining orthologs. Each corrected sequence was constructed from translated genomic sequences (obtained from Ensembl and NCBI databases) after careful examination of alignments performed with either the human amino acid sequence or the amino acid sequence deduced from mRNA sequence when available, or the closed ortholog amino acid sequences. This has been done with particular caution, to respect the integrity of splice site consensus sequences. The phylogenetic tree was drawn with the ClustalX 1.81 and the NJplot programs (1, 2). The human TXNDC3 exon 7 and the TXNDC6 exon 5 were aligned with ClustalX 1.81.
To align the residues encoded by the human TXNDC3 exon 7 and the corresponding exons from Ciona intestinalis to the chimpanzee, we first extracted them from the databases. To this end, we determined the genomic structure of all these genes through alignment of their mRNA sequences when available, or protein sequences from Ensembl database with genomic sequences from either Ensembl or NCBI websites. The location of each exon was determined either manually on the basis of a strict conservation of splice sites or with the use of the model maker tool from the NCBI website, or the FGENESH program at the Softberry website. The accession numbers for protein sequences are the same as those described above. Accession numbers for the human, mouse, rat, and C.intestinalis mRNA sequences are detailed in SI Table 2. For the zebrafish, only a partial sequence of the Txndc3 gene is available in the NCBI database. However, as this sequence includes the region sharing high conservation with human TXNDC3 exon 7, it allowed us to determine the genomic structure of this part of the zebrafish Txndc3 gene. Once the genomic structure of the genes was established, we translated the human exon 7 and the corresponding exon from each species. The translation sequences were then aligned by using the ClustalW 1.81 program.
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