Kumiko Ui-Tei

Short-Interfering-RNA-Mediated Gene Silencing in Mammalian Cells Requires Dicer and eIF2C Translation Initiation Factors

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target.

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO‐K1) as well, although double‐stranded RNA required for the latter was 2500 times more than for the former. Cognate double‐stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.

Formation of the male-specific muscle in female Drosophila by ectopic fruitless expression

The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex-determination factor that directs the development of at least two male-specific characteristics, namely courtship behaviour and formation of the muscle of Lawrence (MOL). The fru gene encodes a putative transcription factor with a BTB domain and two zinc-finger motifs, and with consensus Tra-binding sequences. The binding of Tra to these sequences results in sex-specific alternative splicing of the fru mRNA, leading to production of the ‘male-type’ or ‘female-type’ Fru protein. We show here that the Fru protein is not detected in the female central nervous system (CNS), despite the similar level of expression of fru mRNA in both male and female CNS. As ectopic expression of both the ‘male-type’ (with the sequence for the amino-terminal extension) and ‘female-type’ (without the sequence for the amino-terminal extension) fru cDNA can induce formation of the MOL in females, the presence or absence of the Fru protein, and not its sex-specific structure, seems to be responsible for the sexually dimorphic actions of the fru gene.

Replacement of midgut epithelium in the greater wax moth, Galleria mellonela, during larval-pupal moult.

Abstract. The epithelium of larval midgut of the greater wax moth, Galleria mellonela, was replaced during the larval-pupal moult. The development of this moth was tentatively divided into 11 stages, from the full-grown larva of last instar to the 4-day-old pupa. The midgut at each stage was observed for (1) overall structure, (2) the position of goblet cells, and (3) the appearance of the yellow body. Light microscopy revealed that cell death in the midgut began in a cocoon-spinning larva (stage II), when pigments in the stemmata started to migrate. Before drastic remodeling started to occur, cytoplasmic projections in the goblet cavities were transformed. The larval midgut changed markedly at stage III, when the pigments left the stemmata. The epithelium of the larval midgut dropped as a whole into the lumen, transforming into the yellow body. Simultaneously, a pupal midgut epithelium developed. Electron microscopy of the columnar cells of a stage III larva showed that microvilli and mitochondria looked normal even though the nucleus with condensed heterochromatin resembled an apoptotic nucleus of vertebrate and higher plant cells. Caspase-3-like protease activity was restricted to the larval midgut and increased in parallel with the formation of the yellow body. The results indicate that the replacement of the larval midgut is facilitated by a typical apoptotic process.

A novel gene derived from developing spinal cords, SCDGF, is a unique member of the PDGF/VEGF family

We isolated a novel gene designated spinal cord‐derived growth factor (SCDGF). Its expression was increased in chick spinal cords with embryonic development and decreased after hatching. The amino acid sequences of chick and human SCDGFs revealed a putative signal sequence followed by a CUB domain and a region homologous to the members of the platelet‐derived growth factor/vascular endothelial growth factor family. Furthermore, human SCDGF secreted from the cells showed a mitogenic activity for 10T1/2 cells in vitro. These results led us to speculate that SCDGF plays an important role in the development of the spinal cord.

The promoter region of the human BUBR1 gene and its expression analysis in lung cancer

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

The expression of SCDGF/PDGF-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system.

We examined the expression patterns of the two homologous genes, spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system. In the spinal cord, SCDGF/PDGF-C/fallotein was expressed in the floor plate at embryonic day (E) 11 and also in the ventricular zone at E16 but not in adult. However, SCDGF-B/PDGF-D was prominently expressed in the adult motoneurons, although faint expression was observed in the ventral ventricular zone at E16. Also in the brain, the expression of SCDGF/PDGF-C/fallotein was more remarkable at E16 than at adult. It was highly expressed in the cortex, pontine area and choroid plexus at E16. Contrary to SCDGF/PDGF-C/fallotein, SCDGF-B/PDGF-D expression was notable in several nuclei at adult.

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system. I. ACh and l-DOPA

To characterize neuronal phenotypes, aminergic neurotransmitters, i.e. acetylcholine (ACh), catecholamine (CA)s and other biogenic amines (serotonin, octopamine), were surveyed in 10 colonial clones from one cell line of Drosophila larval CNS using HPLC-ECD system. ACh, which is a neurotransmitter in Drosophila, was found in 7 out of 10 clones. CAs, their metabolites, and other amines were not detected in any clones. However, all the 10 clones expressed L-dopa, a precursor of CAs. Consequently, seven clones expressed ACh and L-dopa. L-dopa as a novel neurotransmitter candidate was discussed.

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca2+ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca2+ ions, although the origin of Ca2+ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca2+ ions whereas those by the other three chemicals utilized intracellular Ca2+ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system. II: Neuropeptides and amino acids

In a previous study, we have found acetylcholine and/or L-DOPA in 10 colonial clones from one cell line of Drosophila larval central nervous system (CNS). In this study, to characterize clonal neuronal phenotypes further, we have examined three neuropeptides and 19 amino acids using HPLC system. Substance P and proctolin were found in seven and eight out of ten clones, respectively. On the other hand, somatostatin was expressed in all ten clones. GABA and taurine were not detected in any clones. Glutamate, which is an excitatory transmitter in Drosophila, was found in all the clones, although its content was different seven times among them. Glycine, which is not known as a transmitter in Drosophila, was found to be unevenly expressed among them. Therefore, the conspicuous expression of peptides or amino acids in some clones suggests that the substances have a special role in Drosophila CNS.