Yasutaka Makino

TIP49b, a New RuvB-like DNA Helicase, Is Included in a Complex Together with Another RuvB-like DNA Helicase, TIP49a

We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB. In this study, we isolated a newTIP49a-related gene, termed TIP49b, from human and yeast cells. TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family. Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase. Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5′ to 3′) was the opposite to that of TIP49a. TIP49b and TIP49a bound to each other and were included in the same complex of ∼700 kDa in a cell. We found thatTIP49b was an essential gene for the growth ofSaccharomyces cerevisiae, as is the TIP49agene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.

A Rat RuvB-like Protein, TIP49a, Is a Germ Cell-enriched Novel DNA Helicase

We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a inEscherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg2+ for the helicase activity were 1–2 mm and 0.25–1 mm, respectively. Displacement of the DNA strand occurred in the 3′ to 5′ direction with respect to the single-stranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.

Spermatid-specific Overexpression of the TATA-binding Protein Gene Involves Recruitment of Two Potent Testis-specific Promoters

The gene encoding the TATA-binding protein, TBP, is highly overexpressed during the haploid stages of spermatogenesis in rodents. RNase protection analyses for mRNAs containing the previously identified first, second, and eighth exons suggested that most TBP mRNAs in testis did not initiate at the first exon used in somatic cells (here designated exon 1C). Using a sensitive ligation-mediated cDNA amplification method, 5′ end variants of TBP mRNA were identified, and the corresponding cDNAs were cloned from liver and testis. In liver, a single promoter/first exon is used to generate a steady-state level of roughly five molecules of TBP mRNA per diploid cell equivalent. In testis, we detect modest up-regulation of the somatic promoter and recruitment of at least five other promoters. Three of the alternative promoter/first exons, including 1C and two of the testis-specific promoter/first exons, 1D and 1E, contribute roughly equivalent amounts of mRNA which, in sum, account for greater than 90% of all TBP mRNA in testis. As a result, round spermatids contain an estimated 1000 TBP mRNA molecules per haploid cell. Testis TBP mRNA also exhibits several low abundance 5′ end splicing variants; however, all detected TBP mRNA leader sequences splice onto the common exon 2 and are expected to initiate translation at the same site within exon 2. The precise locations of the three major initiation exons are mapped on the gene. The identification of the strong testis-specific promoter/first exons will be important for understanding spermatid-specific tbp gene regulation.

Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA-binding protein and a novel transcriptional activator, TIP120.

SUG1 belongs to proteasomal ATPase. Previous studies have demonstrated that SUG1 is associated with TBP. It is assumed to be involved in transcriptional regulation in addition to proteolysis. In this study, we investigated the association of mammalian SUG1 with TBP in more detail.

TATA-Binding Protein-Interacting Protein 120, TIP120, Stimulates Three Classes of Eukaryotic Transcription via a Unique Mechanism

ABSTRACT We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

Association of the rat heterogeneous nuclear RNA-ribonucleoprotein F with TATA-binding protein

Heterogeneous nuclear ribonucleoprotein F (hnRNP‐F) has been shown to be a pre‐mRNA splicing factor. Recent studies have uncovered the coordination of synthesis of pre‐mRNA and its processing, including post‐transcriptional modification and splicing. Here, we present evidence for an association between a splicing factor, hnRNP‐F, and TATA‐binding protein (TBP), which is an essential factor needed for transcription initiation. An affinity detection experiment revealed hnRNP‐F in the preparation of TBP‐interacting proteins. HnRNP‐F was associated with TBP in nuclear extracts and was capable of direct binding to TBP in vitro. These results suggest that hnRNP‐F is associated with TBP in the cell. HnRNP‐F was observed in abundance in the thymus, spleen and testis, and its distribution pattern was similar to that of TBP, implying a functional coordination of transcription and splicing. We assume that the splicing machinery is associated with the transcription apparatus as a prerequisite prior to transcriptional elongation.

Distribution of AP-2 subtypes in the adult mouse brain

In the mammalian central nervous system (CNS), transcription factor activator protein 2 (AP-2) is one of the critical regulatory factors for neural gene expression and neural development. As AP-2 has diverged into several subtypes, i.e. AP-2alpha, -2beta, and 2.2, we investigated the distribution of the AP-2 subtypes in the adult mouse brain by in situ hybridization using subtype-specific probes. Though AP-2 was essentially expressed in most regions of the brain, the hippocampus and cerebellum Purkinje cells exhibited a relatively high concentration of transcripts of any of the AP-2 subtypes. Among AP-2alpha variants, the expression of variant 1 was considerably lower than that of variant 3. Hence, the expression pattern of AP-2alpha variant 3 is suggested to represent the major gene expression of AP-2alpha. On the other hand, the expression of AP-2beta messenger RNA (mRNA) was higher than that of AP-2alpha in many regions. Especially, the olfactory bulb, hippocampus, cerebellum, and cerebral cortex contained an abundance of these mRNAs. Different from those of AP-2alpha, AP-2beta mRNAs were detected in considerable amounts in the glanular cells as well as in Purkinje cells. AP-2.2 gene expression was weak throughout the brain. Consequently, we found that various AP-2 subtypes and variants were expressed in a similar distribution pattern with each having its own specific intensity but that their precise distribution profiles were not exactly the same. In the mature brain, AP-2 is thought to regulate neural gene expression through specific and redundant association with a target gene.

Heterogeneous nuclear RNA‐ribonucleoprotein F binds to DNA via an oligo(dG)‐motif and is associated with RNA polymerase II

The heterogeneous nuclear ribonucleoprotein F (hnRNP‐F) is one of the constituents of the splicing‐related hnRNP complex. Recent studies suggest that pre‐mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre‐mRNA‐engaged factors might be associated with a promoter.

A serine residue in the N-terminal acidic region of rat RPB6, one of the common subunits of RNA polymerases, is exclusively phosphorylated by casein kinase II in vitro ☆

RPB6 is one of the common subunits of all eukaryotic RNA polymerases and is indispensable for the enzyme function. Here, we isolated a rat cDNA encoding RPB6. It contained 127 amino acid (a.a.) residues. From alignment of RPB6 homologues of various eukaryotes, we defined two conserved regions, i.e. an N-terminal acidic region and a C-terminal core. In this study, we investigated in vitro phosphorylation of rat RPB6 by casein kinase II (CKII), a pleiotropic regulator of numerous cellular proteins. Three putative CKII-phosphorylated a.a. within rat RPB6 were assigned. We found that serines were phosphorylated by CKII in vitro. Mutagenesis studies provided evidence that a serine at a.a. position 2 was exclusively phosphorylated. Finally, an RPB6-engaged in-gel kinase assay clarified that CKII was a prominent protein kinase in rat liver nuclear extract that phosphorylates RPB6. Therefore, RPB6 was implied to be phosphorylated by CKII in the nucleus. We postulate that the N-terminal acidic region of the RPB6 subunit has some phosphorylation-coupled regulatory functions.

Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.

Abstract: The type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box‐I element, which is located —334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E‐box consensus sequence, is involved in the up‐regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS‐related basic helix‐loop‐helix (bHLH) factors bind to the box‐I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS‐related bHLH factors, neuronal differentiation factor (NeuroD)‐related factor (NDRF), specifically bound to the box‐I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co‐expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue‐specific regulation of IP3R1 gene expression in the CNS.