Katsuyuki Hashimoto

A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG

Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue‐specific RNA‐binding protein, Fox‐1, which regulates alternative splicing in vertebrates. Fox‐1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox‐1 is expressed in heart and skeletal muscles. As candidates for muscle‐specific targets of Fox‐1, we considered two genes, the human mitochondrial ATP synthase γ‐subunit gene (F1γ) and the rat α‐actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox‐1 induced muscle‐specific exon skipping of the F1γ gene via binding to GCAUG sequences upstream of the regulated exon. Fox‐1 also regulated mutually exclusive splicing of the α‐actinin gene, antagonizing the repressive effect of polypyrimidine tract‐binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox‐1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox‐1 plays key roles in both positive and negative regulation of tissue‐specific splicing via GCAUG.

Rate of Evolution in Brain-Expressed Genes in Humans and Other Primates

Brain-expressed genes are known to evolve slowly in mammals. Nevertheless, since brains of higher primates have evolved rapidly, one might expect acceleration in DNA sequence evolution in their brain-expressed genes. In this study, we carried out full-length cDNA sequencing on the brain transcriptome of an Old World monkey (OWM) and then conducted three-way comparisons among (i) mouse, OWM, and human, and (ii) OWM, chimpanzee, and human. Although brain-expressed genes indeed appear to evolve more rapidly in species with more advanced brains (apes > OWM > mouse), a similar lineage effect is observable for most other genes. The broad inclusion of genes in the reference set to represent the genomic average is therefore critical to this type of analysis. Calibrated against the genomic average, the rate of evolution among brain-expressed genes is probably lower (or at most equal) in humans than in chimpanzee and OWM. Interestingly, the trend of slow evolution in coding sequence is no less pronounced among brain-specific genes, vis-à-vis brain-expressed genes in general. The human brain may thus differ from those of our close relatives in two opposite directions: (i) faster evolution in gene expression, and (ii) a likely slowdown in the evolution of protein sequences. Possible explanations and hypotheses are discussed.

Regulation of constitutive expression of mouse PTEN by the 5'-untranslated region.

PTEN tumor suppressor serves as a major negative regulator of survival signaling mediated by PI3 kinase/AKT/protein kinase B pathway, and is inactivated in various human tumors. Elucidation of mechanisms responsible for PTEN expression is important for providing insight into strategies to control the loss of PTEN expression in human cancers. Although recent studies suggested that p53 and Egr-1 can modulate induced PTEN expression, the mechanism responsible for ubiquitous constitutive expression of PTEN remains elusive. PTEN mRNA contains a highly conserved and GC-rich 5′-untranslated region (5′-UTR). Recently, it has been shown that the long 5′-UTR sequences of several growth-regulated mRNAs contain promoters that can generate mRNAs with shorter 5′-UTRs. In this paper, we tested whether the 5′-UTR sequence of mouse PTEN contains a promoter that is responsible for constitutive expression of PTEN. We found that the long 5′-UTR sequence of mouse PTEN severely inhibits translation of PTEN and a heterologous gene firefly luciferase. Deletion of the most 5′-UTR sequence would enhance translation efficiency 100-fold. We also showed that the 5′-UTR sequence of mouse PTEN does not have an internal ribosome entry site (IRES) that can mediate cap-independent initiation of translation. Instead, we found that the 5′-UTR sequence of mouse PTEN contains a strong promoter that drives the production of a transcript with shorter 5′-UTRs, which can be translated with higher efficiency. This promoter was mapped to the region between −551 and −220 bases upstream of the translation start codon. Cotransfection analysis using Drosophila SL2 cells showed that Sp1 is one of the major transcription factors that can constitutively activate this promoter. Two endogenous PTEN transcripts with 5′-UTRs of 193 and 109 bases were found in DU145 and H226 cell lines. Based on these observations, we conclude that the PTEN expression may be regulated at both transcriptional and translational levels, and that the 5′-UTR sequence of PTEN contains a promoter that is responsible for constitutive PTEN expression.

In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimers disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimers disease.

Evola : Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees

Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliable orthology information is prerequisite for further studies. However, detection of orthologs could be erroneous if pairwise distance-based methods, such as reciprocal BLAST searches, are utilized. Thus, as a sub-database of H-InvDB, an integrated database of annotated human genes (http://h-invitational.jp/), we constructed a fully curated database of evolutionary features of human genes, called ‘Evola’. In the process of the ortholog detection, computational analysis based on conserved genome synteny and transcript sequence similarity was followed by manual curation by researchers examining phylogenetic trees. In total, 18 968 human genes have orthologs among 11 vertebrates (chimpanzee, mouse, cow, chicken, zebrafish, etc.), either computationally detected or manually curated orthologs. Evola provides amino acid sequence alignments and phylogenetic trees of orthologs and homologs. In ‘dN/dS view’, natural selection on genes can be analyzed between human and other species. In ‘Locus maps’, all transcript variants and their exon/intron structures can be compared among orthologous gene loci. We expect the Evola to serve as a comprehensive and reliable database to be utilized in comparative analyses for obtaining new knowledge about human genes. Evola is available at http://www.h-invitational.jp/evola/.

Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta

BackgroundCynomolgus macaques (Macaca fascicularis) are widely used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as rhesus macaques (M. mulatta). We isolated 85,721 clones and determined 9407 full-insert sequences from cynomolgus monkey brain, testis, and liver. These sequences were annotated based on homology to human genes and stored in a database, QFbase http://genebank.nibio.go.jp/qfbase/.ResultsWe found that 1024 transcripts did not represent any public human cDNA sequence and examined their expression using M. fascicularis oligonucleotide microarrays. Significant expression was detected for 544 (51%) of the unidentified transcripts. Moreover, we identified 226 genes containing exon alterations in the untranslated regions of the macaque transcripts, despite the highly conserved structure of the coding regions. Considering the polymorphism in the common ancestor of cynomolgus and rhesus macaques and the rate of PCR errors, the divergence time between the two species was estimated to be around 0.9 million years ago.ConclusionTranscript data from Old World monkeys provide a means not only to determine the evolutionary difference between human and non-human primates but also to unveil hidden transcripts in the human genome. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.

An Alu-linked Repetitive Sequence Corresponding to 280 Amino Acids Is Expressed in a Novel Bovine Protein, but Not in Its Human Homologue

A novel protein harboring a 280-amino acid region from an Alu-linked repetitive sequence (bovine Alu-like dimer-driven family) was isolated from a bovine brain S-100 fraction using monoclonal antibodies against a rat GTPase-activating protein that shares the same epitope. The protein has an apparent molecular mass of 97 kDa (p97). Western blot analysis using extracts prepared from various tissues showed p97 to be predominantly detected in brain and moderately in liver and lung. From sequence analysis of the cDNA encoding p97, it was found that the 840-base pair sequence homologous to a part of the bovine Alu-like dimer-driven family, which has never been shown to be expressed, occurs in the middle of the protein coding region. The protein also contains a pair of intramolecular repeats composed of 40 highly hydrophilic amino acids at the C terminus. Human cDNA homologous to p97 was cloned, and its nucleotide sequence demonstrates that the 840-base pair repetitive sequence and one of the intramolecular repeats are missing. We named p97 bovine BCNT after Bucentaur. These results show that bovine BCNT is a unique molecule and suggest that an analysis of the relationship between bovine bcnt and its human homologue may help further the understanding of gene organization and evolution.

Expressed sequence tags from cynomolgus monkey (Macaca fascicularis) liver: A systematic identification of drug-metabolizing enzymes

The liver, a major organ for drug metabolism, is physiologically similar between monkeys and humans. However, the paucity of identified genes has hampered a deep understanding of drug metabolism in monkeys. To provide such a genetic resource, 28 655 expressed sequence tags (ESTs) were generated from a cynomolgus monkey liver full‐length enriched cDNA library, which contained 23 unique ESTs homologous to human drug‐metabolizing enzymes. Our comparative genomics approach identified nine lineage‐specific candidate ESTs, including three drug‐metabolizing enzymes, which could be important for understanding the physiological differences between monkeys and humans.

MacroH2A1.2 binds the nuclear protein Spop

X-chromosome inactivation is a phenomenon by which one of the two X chromosomes in somatic cells of female mammals is inactivated for life. The inactivated X chromosomes are covered with Xist (X-inactive specific transcript) RNA, and also enriched with the histone H2A variant, macroH2A1.2. The N-terminal one-third of macroH2A1.2 is homologous to core histone H2A, but the function of the C-terminal two-thirds, which contains a basic, putative leucine zipper domain, remains unknown. In this study, we tried analyzing protein-protein interaction with a yeast two-hybrid system to interact with the nonhistone region of mouse macroH2A1.2. The results showed that macroH2A1.2 interacts with mouse nuclear speckled type protein Spop. The Spop protein has a unique composition: an N-terminal MATH, and a C-terminal BTB/POZ domain. Further binding domain mapping in a glutathione-S-transferase (GST) pull-down experiment revealed that macroH2A1.2 binds the MATH domain of Spop, which in turn binds to the putative leucine zipper domain of macroH2A1.2.

Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence

BackgroundIn order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues.ResultThe full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl.We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames) to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously.ConclusionsIn this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.