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Functional implications of C-terminus of TBX5 with high homology to C-terminal domain of yeast DNA-directed RNA polymerase II largest subunit

2008; Lippincott Williams & Wilkins; Volume: 121; Issue: 8 Linguagem: Inglês

10.1097/00029330-200804020-00020

2542-5641

Zhuren Zhou, Li-Guo Gong, Wenqing Geng, Qiu Guang-rong, Kailai Sun,

RNA Research and Splicing

TBX5, as a member of the T-box-containing transcription factor family, encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues.1 The coding region of TBX5 cDNA is 1.5 kb with eight exons including the N-terminal portion, the DNA binding domain and C-terminal region. We reported that the abnormality in transcription level of the TBX5 gene might be the mechanism underlying human simple congenital heart disease in the absence of TBX5 mutations.2 So far, over 37 mutations in the TBX5 gene have been identified in patients with Holt-Oram syndrome,3 an autosomal dominant condition characterized by congenital cardiac malformations and upper limb anomalies. G80R, R237Q and R237W mutations reduce the TBX5 DNA-binding ability with target genes, leading to decreased transcriptional activation of TBX5 target genes and a loss of synergistic transcriptional activation with NKx2-5.4 In contrast, the molecular mechanism by which most TBX5 mutations at the C-terminal region cause Holt-Oram syndrome remains unclear. Moreover, the mechanism underlying the transactivation by the C-terminus of TBX5 is largely unknown. Thus, the sequence-based study of the C-terminus of TBX5 is needed for a better understanding of the molecular mechanism underlying TBX5 mutation-related Holt-Oram syndrome and of TBX5 function as a transcriptional activator. Here, we show that residues 267-448 at the C-terminus of TBX5 are highly homologous to the C-terminal domain (CTD) of yeast DNA-directed RNA polymerase II largest subunit. Our findings have many functional implications for the C-terminus of TBX5 based on the homology and the potential phosphorylation at the C-terminus of TBX5. METHODS TBX5 protein homology search PSI-BLAST5 searches of the non-redundant database were carried out at the NCBI website, using the partial C-terminus of TBX5 (residues 245-448, gi|2920821) as a query. All parameters, including 0.005 as PSI-BLAST threshold, were default. The above C-terminus of TBX5 was also submitted to the FFAS03 server6 (http://ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl) for profile-profile sequence alignments. Furthermore, this sequence was submitted to the HHpred server7 (http://toolkit.tuebingen.mpg.de/hhpred) for a pairwise comparison of Hidden Markov models in the TBX5 protein homology search. Multiple alignments from different species The multiple alignments between residues 267-448 at the C-terminus of TBX5 from a variety of species and the corresponding residues at the CTD of RNA polymerase II were done by M-coffee.8 The species in the alignment were Human, Monkey, Dog, Rat, Mouse, Duckmole, Chicken, Eastern newt, Xenopus laevis and Zebrafish. Phosphorylation site prediction For residues 267-448 at C-terminus of TBX5, prediction of phosphorylation sites was done by PredPhospho,9 which applies support vector machines to predict phosphorylation sites and their corresponding kinases. RESULTS Homology between C-terminus of TBX5 and CTD of yeast DNA-directed RNA polymerase II largest subunit Several iterations of PSI-BLAST searches detected a variety of DNA-directed RNA polymerase II from different species as significant hits to the C-terminus of TBX5. To further explore any potential homology of TBX5, residues 245-448 at the C-terminus of TBX5 were submitted to the FFAS03 and HHpred server. By profile-profile alignment in the FFAS03 sever, residues 245-448 at the C-terminus of TBX5 were highly homologous to DNA-directed RNA polymerase II largest subunit (yeast, NCBI accession: P04050). The FFAS03 score was -29.000 (far below the score-9.5 for significance) and the false positive rate of this prediction was less than 3%. HHpred search results showed that the homology between the C-terminus of TBX5 and DNA-directed RNA polymerase II largest subunit (yeast, NCBI accession: P04050) was statistically significant. The probability for the first hit was 99.8% with E-value 1.8×10-22, and 99.8% with E-value 1.1×10-21 for the second hit. It was notable that the above FFAS03 and HHpred searches identified the same protein yeast DNA-directed RNA polymerase II largest subunit as the significant hit. Well-alignment between C-terminus of TBX5 from different species and CTD of yeast DNA-directed RNA polymerase II largest subunit As shown in Figure, multiple functionally important serine, tyrosine and proline residues at the C-terminus of TBX5 from different species, namely, mammalia (represented by Human, Monkey, Dog, Rat, Mouse, Duckmole), aves (represented by Chicken), amphibia (represented by Eastern newt and Xenopus laevis) and pisces (represented by Zebrafish) were highly conserved and aligned identically with the corresponding consensus heptapeptide repeats at the CTD of RNA polymerase II largest subunit. Moreover, several other serine, tyrosine, proline and threonine residues in the alignment were conserved or semi-conserved, as illustrated by symbols in Figure. As TBX5-related protein sequences for reptilia (for example, lizard and alligator) are not available in the protein databank at present, they were not included in the alignment.Figure.: Multiple alignments between C-terminus of TBX5 from different species and CTD of yeast DNA-directed RNA polymerase II largest subunit by M-coffee. The Tyr291 and Tyr324 residue at C-terminus of TBX5 are indicated with an arrow and an arrowhead, respectively. *The residues in that column were identical in all sequences in the alignment;: Conserved substitutions were observed, although residues in that column were not identical in all sequences in the alignment;. Semi-conserved substitutions were observed, although residues in that column were not identical in all sequences in the alignment.Tyr291 and Tyr342 at the C-terminus of TBX5 as potential phosphorylation sites The potential phosphorylation sites at the C-terminus of TBX5 were predicted by PredPhospho.9 Both Tyr291 and Tyr342 residues may undergo phosphorylation by tyrosine kinase. DISCUSSION After the completion of the Human Genome Project by an international cooperation, vast information about protein sequences is open to the public and await further analysis. Powerful bioinformatic tools emerged as an alternative way for protein function research, without resorting to the traditional, labor-consuming experimentation.10 PSI-BLAST is proven to be a useful tool for inferring protein function from sequence relationships. But such profile-sequence based methods sometimes are not sensitive enough to detect distant homology for the protein of interest. Several profile-profile comparison based programs have been developed for remote homology inference, such as FFAS03 and HHpred. FFAS03 is a profile-profile comparison server which enables detection of remote homology. HHpred, as a fast server with the highest sensitivity and alignment accuracy for remote protein homology detection and structure prediction, exploits pairwise comparison of profile Hidden Markov models. In this paper, our results by PSI-BLAST, FFAS03 and HHpred with distinct algorithms, showed that residues 267-448 at the C-terminus of TBX5 are highly homologous to the CTD of yeast DNA-directed RNA polymerase II largest subunit. The CTD of DNA-directed RNA polymerase II largest subunit consists of a set of tandemly repeated, evolutionarily conserved heptapeptides (Y1-S2-P3-T4-S5-P6-S7), with residues S2 and S5 being the predominant sites of phosphorylation. Y1 is also a site of phosphorylation in mammalian cells and its phosphorylation can be catalyzed by either c-Abl or Arg kinase.11 Our findings of the high homology between the C-terminus of TBX5 with the CTD of RNA polymerae II largest subunit have many implications for the TBX5 function. First, the highly-conserved C-terminus of TBX5 from different species and their well-alignment with the consensus heptapeptide repeats at the CTD of RNA polymerase II demonstrate several functionally important sites for further studies. Among them, the Tyr291 and Tyr342 residues deserve our attention. We found that both residues were highly conserved among species and identical with the corresponding tyrosine, the first residue in consensus repeated heptapeptides at the CTD of RNA polymerase II. Meanwhile, they were potential phosphorylation sites predicted by PredPhospho. Furthermore, mutations at residues Tyr291 and Tyr342 at the C-terminus of TBX5 were reported to be related to Holt-Oram syndrome.3 Given the fact that phosphorylation is a ubiquitous cellular regulatory mechanism for the normal function of a protein and phosphorylation at Thr278 were recently reported at mouse Tbx5,12 we may add here that loss of phosphorylation sites, if any, on Tyr291 and Tyr342 may also contribute to the molecular mechanism underlying Holt-Oram syndrome with Tyr291 or Tyr342 mutation. Second, a feature common to the C-terminus of TBX5 and CTD of RNA polymerase II is that they are all capable of interacting with and recruiting other proteins. The C-terminal region of TBX5 forms the structure through which TBX5 interacts with its potent coactivator TAZ, a WW-domain containing a transcriptional regulator.13 Interestingly, phosphorylation on CTD fragments of RNA polymerase II could lead to the tight binding of RNA polymerase II to the WW domain-containing protein.14 Studies also provide in vivo significance of the LMP4/Tbx5 protein interaction for the Tbx5 to function well in both the nucleus and cytoplasm.15 Similarly, by interacting with distinct proteins, CTD plays a pivotal role in just about all transcription reaction. Meanwhile, our study showed the high homology between the C-terminus of TBX5 and the CTD of DNA-directed RNA polymerase II largest subunit. Taking all these into consideration, we propose that the partial function of the C-terminus of TBX5 is to interact with or recruit other proteins for its transcriptional activity. In summary, we show that residues 267-448 at C-terminus of TBX5 are highly homologous to the yeast CTD of RNA polymerase II largest subunit. Highly-conserved Tyr291 and Tyr342 residues at the C-terminus of TBX5 are potential phosphorylation sites. The C-terminus of TBX5 may play the role in transcriptional activity by interacting with or recruiting other proteins. The patterns of phosphorylation at the C-terminus of TBX5 deserve further investigations to explore the mechanism underlying TBX5 mutation-related Holt-Oram syndrome and TBX5 transcriptional activity.