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1.  Structural and functional analyses of Barth syndrome-causing mutations and alternative splicing in the tafazzin acyltransferase domain 
Meta Gene  2015;4:92-106.
Tafazzin is a mitochondrial phospholipid transacylase, and its mutations cause Barth syndrome (BTHS). Human tafazzin gene produces four distinct alternatively spliced transcripts. To understand the molecular mechanisms of tafazzin deficiency, we performed an atomic resolution analysis of the influence of the BTHS mutations and of alternative splicing on the structure and function of tafazzin. From the three-dimensional (3D) homology modeling of tafazzin, we identified candidate amino acid residues that contribute to cardiolipin binding and to mitochondrial membrane associations that facilitate acyl-transfer reactions. Primate specific exon 5, which is alternatively spliced, is predicted to correspond to an intrinsically unstructured region in the protein. We proposed that this region should change the substrate-binding affinity and/or contribute to primate-specific molecular interactions. Exon 7, another alternatively spliced exon, encodes a region forming a part of the putative substrate-binding cleft, suggesting that the gene products lacking exon 7 will lose their substrate-binding ability. We demonstrate a clear localization of the BTHS mutations at residues responsible for membrane association, substrate binding, and the conformational stability of tafazzin. These findings provide new insights into the function of defective tafazzin and the pathogenesis of BTHS at the level of protein 3D structure and the evolution of alternatively spliced exons in primates.
•We predicted a 3D structure model of tafazzin with reasonable reliability.•The model structure accounts for differences in function of splice variants.•The exon acquired in the primate lineage encodes an intrinsically unstructured region.•The mutations associated with Barth syndrome were found on functionally important residues of tafazzin.
PMCID: PMC4412953  PMID: 25941633
TAZ gene; X-linked recessive disease; Disease-causing mutations; Homology modeling; Immunodeficiency; Intrinsically unstructured region
2.  AS-EAST: a functional annotation tool for putative proteins encoded by alternatively spliced transcripts 
Bioinformatics  2012;28(15):2076-2077.
Summary: Alternative Splicing Effects ASsessment Tools (AS-EAST) is an online tool for the functional annotation of putative proteins encoded by transcripts generated by alternative splicing (AS). When provided with a transcript sequence, AS-EAST identifies regions altered by AS events in the putative protein sequence encoded by the transcript. Users can evaluate the predicted function of the putative protein by inspecting whether functional domains are included in the altered regions. Moreover, users can infer the loss of inter-molecular interactions in the protein network according to whether the AS events affect interaction residues observed in the 3D structure of the reference isoform. The information obtained from AS-EAST will help to design experimental analyses for the functional significance of novel splice isoforms.
Availability: The online tool is freely available at
PMCID: PMC3400965  PMID: 22645168
3.  RESOPS: A Database for Analyzing the Correspondence of RNA Editing Sites to Protein Three-Dimensional Structures 
Plant and Cell Physiology  2009;50(11):1865-1873.
Transcripts from mitochondrial and chloroplast DNA of land plants often undergo cytidine to uridine conversion-type RNA editing events. RESOPS is a newly built database that specializes in displaying RNA editing sites of land plant organelles on protein three-dimensional (3D) structures to help elucidate the mechanisms of RNA editing for gene expression regulation. RESOPS contains the following information: unedited and edited cDNA sequences with notes for the target nucleotides of RNA editing, conceptual translation from the edited cDNA sequence in pseudo-UniProt format, a list of proteins under the influence of RNA editing, multiple amino acid sequence alignments of edited proteins, the location of amino acid residues coded by codons under the influence of RNA editing in protein 3D structures and the statistics of biased distributions of the edited residues with respect to protein structures. Most of the data processing procedures are automated; hence, it is easy to keep abreast of updated genome and protein 3D structural data. In the RESOPS database, we clarified that the locations of residues switched by RNA editing are significantly biased to protein structural cores. The integration of different types of data in the database also help advance the understanding of RNA editing mechanisms. RESOPS is accessible at
PMCID: PMC2775959  PMID: 19808808
Chloroplast; Mitochondrion; Molecular evolution; Organelle genome; Protein 3D structure; RNA editing
4.  AS-ALPS: a database for analyzing the effects of alternative splicing on protein structure, interaction and network in human and mouse 
Nucleic Acids Research  2008;37(Database issue):D305-D309.
We have constructed a database, AS-ALPS (alternative splicing-induced alteration of protein structure), which provides information that would be useful for analyzing the effects of alternative splicing (AS) on protein structure, interactions with other bio-molecules and protein interaction networks in human and mouse. Several AS events have been revealed to contribute to the diversification of protein structure, which results in diversification of interaction partners or affinities, which in turn contributes to regulation of bio-molecular networks. Most AS variants, however, are only known at the sequence level. It is important to determine the effects of AS on protein structure and interaction, and to provide candidates for experimental targets that are relevant to network regulation by AS. For this purpose, the three-dimensional (3D) structures of proteins are valuable sources of information; however, these have not been fully exploited in any other AS-related databases. AS-ALPS is the only AS-related database that describes the spatial relationships between protein regions altered by AS (‘AS regions’) and both the proteins’ hydrophobic cores and sites of inter-molecular interactions. This information makes it possible to infer whether protein structural stability and/or protein interaction are affected by each AS event. AS-ALPS can be freely accessed at and
PMCID: PMC2686549  PMID: 19015123
5.  Correlation between amino acid residues converted by RNA editing and functional residues in protein three-dimensional structures in plant organelles 
BMC Plant Biology  2008;8:79.
In plant organelles, specific messenger RNAs (mRNAs) are subjected to conversion editing, a process that often converts the first or second nucleotide of a codon and hence the encoded amino acid. No systematic patterns in converted sites were found on mRNAs, and the converted sites rarely encoded residues located at the active sites of proteins. The role and origin of RNA editing in plant organelles remain to be elucidated.
Here we study the relationship between amino acid residues encoded by edited codons and the structural characteristics of these residues within proteins, e.g., in protein-protein interfaces, elements of secondary structure, or protein structural cores. We find that the residues encoded by edited codons are significantly biased toward involvement in helices and protein structural cores. RNA editing can convert codons for hydrophilic to hydrophobic amino acids. Hence, only the edited form of an mRNA can be translated into a polypeptide with helix-preferring and core-forming residues at the appropriate positions, which is often required for a protein to form a functional three-dimensional (3D) structure.
We have performed a novel analysis of the location of residues affected by RNA editing in proteins in plant organelles. This study documents that RNA editing sites are often found in positions important for 3D structure formation. Without RNA editing, protein folding will not occur properly, thus affecting gene expression. We suggest that RNA editing may have conferring evolutionary advantage by acting as a mechanism to reduce susceptibility to DNA damage by allowing the increase in GC content in DNA while maintaining RNA codons essential to encode residues required for protein folding and activity.
PMCID: PMC2488346  PMID: 18631376
6.  Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs 
Nucleic Acids Research  2006;34(14):3917-3928.
We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.
PMCID: PMC1557807  PMID: 16914452
7.  Coverage of whole proteome by structural genomics observed through protein homology modeling database 
We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (, which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics.
PMCID: PMC1769342  PMID: 17146617
domain duplication; domain interactions; genome; homology modeling; P-loop; structural genomics
8.  Enlarged FAMSBASE: protein 3D structure models of genome sequences for 41 species 
Nucleic Acids Research  2003;31(1):463-468.
Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System (FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF (open reading frame), ORF keywords, Protein Data Bank (PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at
PMCID: PMC165564  PMID: 12520053
9.  Revisiting gap locations in amino acid sequence alignments and a proposal for a method to improve them by introducing solvent accessibility 
Proteins  2011;79(6):1868-1877.
In comparative modeling, the quality of amino acid sequence alignment still constitutes a major bottleneck in the generation of high quality models of protein three-dimensional (3D) structures. Substantial efforts have been made to improve alignment quality by revising the substitution matrix, introducing multiple sequences, replacing dynamic programming with hidden Markov models, and incorporating 3D structure information. Improvements in the gap penalty have not been a major focus, however, following the development of the affine gap penalty and of the secondary structure dependent gap penalty. We revisited the correlation between protein 3D structure and gap location in a large protein 3D structure data set, and found that the frequency of gap locations approximated to an exponential function of the solvent accessibility of the inserted residues. The nonlinearity of the gap frequency as a function of accessibility corresponded well to the relationship between residue mutation pattern and residue accessibility. By introducing this relationship into the gap penalty calculation for pairwise alignment between template and target amino acid sequences, we were able to obtain a sequence alignment much closer to the structural alignment. The quality of the alignments was substantially improved on a pair of sequences with identity in the “twilight zone” between 20 and 40%. The relocation of gaps by our new method made a significant improvement in comparative modeling, exemplified here by the Bacillus subtilis yitF protein. The method was implemented in a computer program, ALAdeGAP (ALignment with Accessibility dependent GAp Penalty), which is available at Proteins 2011; © 2011 Wiley-Liss, Inc.
PMCID: PMC3110861  PMID: 21465562
ALAdeGAP; amino acid sequence alignment; comparative modeling; position dependent gap penalty; solvent accessibility
10.  Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones 
Imanishi, Tadashi | Itoh, Takeshi | Suzuki, Yutaka | O'Donovan, Claire | Fukuchi, Satoshi | Koyanagi, Kanako O | Barrero, Roberto A | Tamura, Takuro | Yamaguchi-Kabata, Yumi | Tanino, Motohiko | Yura, Kei | Miyazaki, Satoru | Ikeo, Kazuho | Homma, Keiichi | Kasprzyk, Arek | Nishikawa, Tetsuo | Hirakawa, Mika | Thierry-Mieg, Jean | Thierry-Mieg, Danielle | Ashurst, Jennifer | Jia, Libin | Nakao, Mitsuteru | Thomas, Michael A | Mulder, Nicola | Karavidopoulou, Youla | Jin, Lihua | Kim, Sangsoo | Yasuda, Tomohiro | Lenhard, Boris | Eveno, Eric | Suzuki, Yoshiyuki | Yamasaki, Chisato | Takeda, Jun-ichi | Gough, Craig | Hilton, Phillip | Fujii, Yasuyuki | Sakai, Hiroaki | Tanaka, Susumu | Amid, Clara | Bellgard, Matthew | de Fatima Bonaldo, Maria | Bono, Hidemasa | Bromberg, Susan K | Brookes, Anthony J | Bruford, Elspeth | Carninci, Piero | Chelala, Claude | Couillault, Christine | de Souza, Sandro J. | Debily, Marie-Anne | Devignes, Marie-Dominique | Dubchak, Inna | Endo, Toshinori | Estreicher, Anne | Eyras, Eduardo | Fukami-Kobayashi, Kaoru | R. Gopinath, Gopal | Graudens, Esther | Hahn, Yoonsoo | Han, Michael | Han, Ze-Guang | Hanada, Kousuke | Hanaoka, Hideki | Harada, Erimi | Hashimoto, Katsuyuki | Hinz, Ursula | Hirai, Momoki | Hishiki, Teruyoshi | Hopkinson, Ian | Imbeaud, Sandrine | Inoko, Hidetoshi | Kanapin, Alexander | Kaneko, Yayoi | Kasukawa, Takeya | Kelso, Janet | Kersey, Paul | Kikuno, Reiko | Kimura, Kouichi | Korn, Bernhard | Kuryshev, Vladimir | Makalowska, Izabela | Makino, Takashi | Mano, Shuhei | Mariage-Samson, Regine | Mashima, Jun | Matsuda, Hideo | Mewes, Hans-Werner | Minoshima, Shinsei | Nagai, Keiichi | Nagasaki, Hideki | Nagata, Naoki | Nigam, Rajni | Ogasawara, Osamu | Ohara, Osamu | Ohtsubo, Masafumi | Okada, Norihiro | Okido, Toshihisa | Oota, Satoshi | Ota, Motonori | Ota, Toshio | Otsuki, Tetsuji | Piatier-Tonneau, Dominique | Poustka, Annemarie | Ren, Shuang-Xi | Saitou, Naruya | Sakai, Katsunaga | Sakamoto, Shigetaka | Sakate, Ryuichi | Schupp, Ingo | Servant, Florence | Sherry, Stephen | Shiba, Rie | Shimizu, Nobuyoshi | Shimoyama, Mary | Simpson, Andrew J | Soares, Bento | Steward, Charles | Suwa, Makiko | Suzuki, Mami | Takahashi, Aiko | Tamiya, Gen | Tanaka, Hiroshi | Taylor, Todd | Terwilliger, Joseph D | Unneberg, Per | Veeramachaneni, Vamsi | Watanabe, Shinya | Wilming, Laurens | Yasuda, Norikazu | Yoo, Hyang-Sook | Stodolsky, Marvin | Makalowski, Wojciech | Go, Mitiko | Nakai, Kenta | Takagi, Toshihisa | Kanehisa, Minoru | Sakaki, Yoshiyuki | Quackenbush, John | Okazaki, Yasushi | Hayashizaki, Yoshihide | Hide, Winston | Chakraborty, Ranajit | Nishikawa, Ken | Sugawara, Hideaki | Tateno, Yoshio | Chen, Zhu | Oishi, Michio | Tonellato, Peter | Apweiler, Rolf | Okubo, Kousaku | Wagner, Lukas | Wiemann, Stefan | Strausberg, Robert L | Isogai, Takao | Auffray, Charles | Nomura, Nobuo | Gojobori, Takashi | Sugano, Sumio
PLoS Biology  2004;2(6):e162.
The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.
An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community
PMCID: PMC393292  PMID: 15103394

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