Akira Ishiguro

RNA Polymerase II Subunits 2, 3, and 11 Form a Core Subassembly with DNA Binding Activity

RNA polymerase II purified from the fission yeastSchizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni2+ affinity chromatography. After stepwise dissociation of the Rpb1- and Rpb3-tagged RNA polymerases fixed on Ni2+-resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6m urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the α2β complex in the assembly of Escherichia coliRNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.

Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

ABSTRACT The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (τ131) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37°C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5′ ends. Maturation of tRNA was found to be aberrant in bdp1-Δ253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1Δ253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.

The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

ABSTRACT The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeastSchizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathioneS-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

ZIC2-dependent Transcriptional Regulation Is Mediated by DNA-dependent Protein Kinase, Poly(ADP-ribose) Polymerase, and RNA Helicase A

The Zic family of zinc finger proteins is essential for animal development, as demonstrated by the holoprosencephaly caused by mammalian Zic2 mutation. To determine the molecular mechanism of Zic-mediated developmental control, we characterized two types of high molecular weight complexes, including Zic2. Complex I was composed of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70/80, and poly(ADP-ribose) polymerase; complex II contained Ku70/80 and RNA helicase A; all the components interacted directly with Zic2 protein. Immunoprecipitation, subnuclear localization, and in vitro phosphorylation analyses revealed that the DNA-PKcs in complex I played an essential role in the assembly of complex II. Stepwise exchange from complex I to complex II depended on phosphorylation of Zic2 by DNA-PK and poly-(ADP-ribose) polymerase. Phosphorylated Zic2 protein made a stable complex with RNA helicase A, and complex II could interact with RNA polymerase II. Phosphorylation-dependent transformation of Zic2-containing molecular complexes may occur in transcriptional regulation.

TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation.

Growth and differentiation of the neurites depends on long‐distance transport of a specific set of mRNAs to restricted area and their local translation. Here, we found that a TAR DNA‐binding protein of 43 kDa in size (TDP‐43) plays an essential role in intracellular transport of mRNA. For identification of target RNAs recognized by TDP‐43, we purified TDP‐43 in soluble dimer form and subjected to in vitro systematic evolution of ligands by exponential enrichment (SELEX) screening. All the TDP‐43‐bound RNAs were found to contain G‐quadruplex (G4). Using a double‐fluorescent probe system, G4‐containing RNAs were found to be transported, together with TDP‐43, into the distal neurites. Two lines of evidence indicated that loss of function of TDP‐43 results in the neurodegenerative disorder: (i) amyotrophic lateral sclerosis (ALS)‐linked mutant TDP‐43M337V lacks the activity of binding and transport of G4‐containing mRNAs; and (ii) RNA containing G4‐forming GGGGCC repeat expansion from the ALS‐linked C9orf72 gene absorbs TDP‐43, thereby reducing the intracellular pool of functional TDP‐43. Taken together, we propose that TDP‐43 within neurons plays an essential role of mRNA transport into distal neurites for local translation, and thus, dysfunctions of TDP‐43 cause neural diseases such as ALS and frontotemporal lobar degeneration.

Rines/RNF180, a novel RING finger gene-encoded product, is a membrane-bound ubiquitin ligase

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled‐coil domain, a novel conserved domain (DSPRC) and a C‐terminal hydrophobic region that is predicted to be a transmembrane domain. N‐terminally epitope tagged‐Rines (Nt‐Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt‐Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt‐Rines is an integral membrane protein with most of its N‐terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt‐Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt‐Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin–proteasome pathway was further supported by its binding to the UbcH6 ubiquitin‐conjugating enzyme and by its trans‐ubiquitination enhancing activities. These results suggest that Rines is a membrane‐bound E3 ubiquitin ligase.

CD spectra show the relational style between Zic-, Gli-, Glis-zinc finger protein and DNA

Zic family proteins have five C2H2-type zinc finger motifs. The Zic-zinc finger domains show high homology to the corresponding domains of the Gli and Glis families, which also contain five C2H2-type zinc finger motifs. The zinc finger motifs of the proteins of these three protein families form an alpha-helix conformation in solution. The addition of oligo DNA that included a Gli-binding sequence increased the alpha-helix content estimated by using circular dichroism spectroscopy. Comparison of the Zic-, Gli-, and Glis-zinc fingers indicated that the alpha-helix content after the addition of oligo DNA correlated well with the affinity of each zinc finger for the oligo DNA (correlation coefficient, 0.85). The importance of the zinc ion for protein folding was reflected in a reduction in the alpha-helix content upon removal of the zinc ion. Owing to the compact globular structure, the alpha-helix structure of the proteins of these three protein families is extremely thermally stable. These results suggest that the alpha-helix structure is important for DNA binding and profoundly related to functional and structural diversity among the three families.

Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients

ZIC2 is a causal gene for holoprosencephaly and encodes a zinc-finger-type transcriptional regulator. We characterized Zic2kd/+ mice with a moderate (40%) reduction in Zic2 expression. Zic2kd/+ mice showed increased locomotor activity in novel environments, cognitive and sensorimotor gating dysfunctions, and social behavioral abnormalities. Zic2kd/+ brain involved enlargement of the lateral ventricle, thinning of the cerebral cortex and corpus callosum, and decreased number of cholinergic neurons in the basal forebrain. Because these features are reminiscent of schizophrenia, we examined ZIC2 variant-carrying allele frequencies in schizophrenia patients and in controls in the Japanese population. Among three novel missense mutations in ZIC2, R409P was only found in schizophrenia patients, and was located in a strongly conserved position of the zinc finger domain. Mouse Zic2 with the corresponding mutation showed lowered transcription-activating capacity and had impaired target DNA-binding and co-factor-binding capacities. These results warrant further study of ZIC2 in the pathogenesis of schizophrenia.

Functional role of Zic2 phosphorylation in transcriptional regulation

Zic2 is a transcriptional activator that plays a crucial role in mammalian forebrain development. It activates the transcription of target genes by DNA binding and recruitment of RNA helicase A (RHA). We recently reported that the Zic2–RHA interaction is decreased by phosphatase treatment in vitro. We have now identified the phosphorylation site (serine 200) in mouse Zic2. Zic2S200A was defective in RHA‐binding, and its transcriptional activation ability was diminished. These data indicate that Zic2S200 is a target for phosphorylation by DNA‐dependent protein kinase, regulating Zic2‐mediated transcriptional activation.

Link between the causative genes of holoprosencephaly: Zic2 directly regulates Tgif1 expression

One of the causal genes for holoprosencephaly (HPE) is ZIC2 (HPE5). It belongs to the zinc finger protein of the cerebellum (Zic) family of genes that share a C2H2-type zinc finger domain, similar to the GLI family of genes. In order to clarify the role of Zic2 in gene regulation, we searched for its direct target genes using chromatin immunoprecipitation (ChIP). We identified TGIF1 (HPE4), another holoprosencephaly-causative gene in humans. We identified Zic2-binding sites (ZBS) on the 5′ flanking region of Tgif1 by in vitro DNA binding assays. ZBS were essential for Zic2-dependent transcriptional activation in reporter gene assays. Zic2 showed a higher affinity to ZBS than GLI-binding sequences. Zic2-binding to the cis-regulatory element near the Tgif1 promoter may be involved in the mechanism underlying forebrain development and incidences of HPE.