RNA interference-based strategies for metabolic syndrome treatment.

RNA interference is a naturally occurring cellular mechanism to inhibit the expression of specific gene products. The technical application of RNA interference offers great potential for the specific treatment of a huge variety of diseases including the metabolic syndrome, one of the most challenging threats to human health associated with our civilization. In order to develop novel and powerful strategies for the treatment of the metabolic syndrome, it is essential to define a set of specific gene products that may be targeted by RNA interference. Based on currently available in vitro and in vivo data, we discuss the feasibility of candidate genes involved in the pathophysiology of the metabolic syndrome as potential targets for a rational RNA interference based therapy in this review.

Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development.

The structural proteins of the cytoplasmic intermediate filaments (IFs) arise in the nematode Caenorhabditis elegans from eight reported genes and an additional three genes now identified in the complete genome. With the use of double-stranded RNA interference (RNAi) for all 11 C. elegans genes encoding cytoplasmic IF proteins, we observe phenotypes for the five genes A1, A2, A3, B1, and C2. These range from embryonic lethality (B1) and embryonic/larval lethality (A3) to larval lethality (A1 and A2) and a mild dumpy phenotype of adults (C2). Phenotypes A2 and A3 involve displaced body muscles and paralysis. They probably arise by reduction of hypodermal IFs that participate in the transmission of force from the muscle cells to the cuticle. The B1 phenotype has multiple morphogenetic defects, and the A1 phenotype is arrested at the L1 stage. Thus, at least four IF genes are essential for C. elegans development. Their RNAi phenotypes are lethal defects due to silencing of single IF genes. In contrast to C. elegans, no IF genes have been identified in the complete Drosophila genome, posing the question of how Drosophila can compensate for the lack of these proteins, which are essential in mammals and C. elegans. We speculate that the lack of IF proteins in Drosophila can be viewed as cytoskeletal alteration in which, for instance, stable microtubules, often arranged as bundles, substitute for cytoplasmic IFs.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Identification of essential genes in cultured mammalian cells using small interfering RNAs.

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.

GAS41, a highly conserved protein in eukaryotic nuclei, binds to NuMA.

The yeast two-hybrid system was used to identify binding partners of NuMA, a component of the nuclear matrix in interphase cells. By using the C-terminal half of NuMA as bait, a human cDNA sequence coding for a 223-amino acid protein with a non-helical N-terminal domain and a C-terminal alpha-helical portion was identified and fully sequenced. It was identical to GAS41, a sequence amplified in human gliomas. The sequence of the homologous Drosophila protein was established, and the alignment for GAS41 from nine different species showed that GAS41 is a general eukaryotic protein found in species as diverse as Arabidopsis, Drosophila, Caenorhabditis elegans, yeast, and man. Northern blot analysis showed a single transcript in eight human tissues. A polyclonal antibody to GAS41 showed a dotted staining pattern in interphase nuclei and a uniform distribution in mitotic cells. A GFP-GAS41 fusion protein displayed equivalent patterns. In vitro GAS41 bound to the C-terminal part of the rod region of NuMA, as shown by dot overlay and by surface plasmon resonance measurements. The K(d) of the complex was 2 x 10(-)(7) m. GAS41 is related to the AF-9 and ENL proteins, which are putative transcription factors found as fusion proteins in some acute leukemias. The NuMA/GAS41 interaction may provide a link between nuclear structure and gene expression.

Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice.

NuMA is a nuclear matrix protein in interphase and relocates to the spindle poles in mitotis. Different NuMA constructs, in which either N- or C-terminal domains were deleted, and the full-length construct were expressed in Escherichia coli, and the NuMA polypeptides were purified to homogeneity and allowed to assemble in vitro. Electron microscopy showed that NuMA can build multiarm oligomers by interaction of the C-terminal globular domains. Each arm of the oligomer corresponds to a NuMA dimer. Oligomers with up to 10 or 12 arms have been observed for both full-length NuMA and for constructs that still contain the proximal part of the C-terminal tail domain. Other results from this laboratory have shown that transient overexpression of NuMA in HeLa cells induces a nuclear scaffold with a quasi-hexagonal organization that can fill the nuclei. Here we show that computer modelling of the three-dimensional packing of NuMA into such scaffolds can explain the different spacing of the hexagons seen when constructs with different coiled-coil lengths are used. Thus, the 12 arm oligomer, for which we have in vitro evidence, may be the structural unit from which the nuclear scaffold in transfected cells is built.

Does NuMA have a scaffold function in the interphase nucleus?

We review the properties of NuMA, concentrating on a possible role for NuMA as a scaffold protein in the interphase nucleus. NuMA is a component of the nuclear matrix in interphase cells and translocates to the spindle poles in mitosis. NuMA has a secondary structure in which a long central rod domain that forms a double-stranded coiled coil is flanked by globular terminal domains. In vitro assembly experiments with bacterially expressed recombinant protein showed that NuMA seems not to form filaments, but instead builds multiarm oligomers by interaction of the C-terminal globular domains. Transient overexpression of NuMA in HeLa cells induced the formation of a three-dimensional lattice with a quasihexagonal organization that fills the nucleus. Use of mutant constructs showed that the lattice spacing depended on the length of the rod domain. Using a 12-arm oligomer as the structural unit, computer modeling can explain the observed nuclear lattices. The flexibility of the NuMA molecule as well as its dynamic capacity to form lattices is a hint that NuMA may play a structural role in the architecture of the normal interphase nucleus.

Induction of a regular nuclear lattice by overexpression of NuMA.

Transient overexpression of nuclear mitotic apparatus protein (NuMA) in HeLa cells results in ordered lattices which can fill the nucleus and which are stable to detergent extraction. Electron microscopy reveals a quasi-hexagonal organization with an average spacing between the vertices of approximately 170 nm and short 6-nm-diameter rods connecting the vertices. Overexpression of a NuMA construct with an in-frame addition in the coiled-coil domain shows hexagons with the spacing increased by 42% while constructs with deletions in the coiled-coil domain yield hexagons with the spacing decreased by 40 and 19%. NuMA constructs truncated at residue 2005 or 2030 in the tail domain cause a drastic reorganization of nuclear components with relocation of the DNA, histone H1, and nucleoli to the nuclear rim. A construct lacking the head and much of the coiled-coil region also affects nuclear organization. In contrast, NuMA constructs truncated at residue 1950 or 1935 which lack the nuclear localization signal display normal nuclear structure but form cytoplasmic aggregates which also display hexagonal organization. Immunoelectron microscopy confirms that the nuclear lattices are built from NuMA. We discuss the importance of the different domains of NuMA for building the ordered in vivo lattices and whether NuMA could play a structural role in the architecture of the normal interphase nucleus.

Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded coiled-coil in the NuMA protein.

NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis. NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation. We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments. The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified. To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity. They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy. The results directly reveal the tripartate structure of NuMA. A long central rod domain is flanked by globular end domains. The rod is 207 nm long and is at least 90% alpha-helical. It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register. The NuMA protein thus forms the longest coiled-coil currently known. Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures.

New monoclonal antibodies recognizing phosphorylated proteins in mitotic cells.

Three monoclonal antibodies which showed strong staining of mitotic cells by screening on the human cell line MCF-7 were isolated. The antigens detected by the DH7 and BF6 monoclonal antibodies were located predominantly in multiple extranucleolar patches in interphase cell nuclei. In mitotic cells a strong increase in the fluorescence intensity was accompanied by its redistribution into a fine speckled form. Metaphase chromosomes were unstained. Centrosomes, spindle poles or midbodies were not stained either before or after extraction of the cells with Triton X-100 under conditions which preserve microtubular structures. In immunoblots of interphase cell extracts only very few bands reacted with DH7 whereas in mitotic cell extracts approximately 30 bands were stained. BF6 also showed an increase in the intensity and number of bands detected in mitotic compared to interphase cell extracts, and the pattern was clearly different from that obtained with DH7. The BF6 antigen were extracted by 0.5% Triton X-100, whereas the DH7 antigen was not. Dephosphorylation of the antigens strongly reduced the binding of both antibodies as measured by immunoblotting and ELISA assays. The results suggested that BF6 and DH7 detect two different phosphorylated epitopes, each of which is shared by a different subset of proteins from mitotic cells. The third antibody, BD 12, bound to several polypeptides, including one of high molecular weight that appeared to correspond to the NuMA antigen. The epitope recognized by BD 12 was not sensitive to phosphatases.

Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells.

Several high molecular mass proteins which relocate from the interphase nucleus to the spindle poles during mitosis have been defined by antibodies. Microinjection experiments have shown that at least the antigen defined by SPN antibody plays a functional role during mitosis. Recently the cDNA sequence for human NuMA antigen was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to be included in the NuMA protein. Here we show that immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage and covering more than fifty amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA are the same protein. Injection of SPN-3 antibody into interphase or mitotic PtK2 cells results in cells with micronuclei. For cells injected in prophase, prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with micronuclei. In contrast only 16% of cells injected in anaphase are abnormal. Thus SPN/NuMA antigen may be required during early, but not during later, stages of mitosis. Surprising parallels are seen between the effects of microinjecting SPN-3 antibody and treatment with colcemid and taxol of PtK2 and HeLa cells. Our results identify an important role during mitosis for the SPN/NuMA antigen.

Studies of the Enzymic Capacities and Transport Properties of Pea Root Plastids.

Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.