Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.     

Use of random ribozyme libraries for the rapid screening of apoptosis- and metastasis-related genes

Apoptosis and metastasis are complex processes involving many gene products. Various biochemical, genetic and technological approaches are used to identify functional gene products, and are valuable for understanding basic biology and disease. However, now that sequence information is abundant and accessible through databases, there is a simpler, more definitive technology to identify genes that are the most relevant to biological phenomena of interest. A rapid screening system using libraries of randomized ribozymes has been developed that identifies key gene products involved in a defined phenotype using a functional gene ‘knock-down’ approach. Libraries of randomized ribozymes have been used to identify genes involved in pathways of apoptosis and metastasis, and there is great potential for this system to identify genes involved in other processes and diseases as well.     

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.     

Inhibition of p38 kinase reveals a TNF-alpha-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line

TNF-alpha transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-kappaB-mediated transactivation, without affecting the nuclear translocation of NF-kappaB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.     

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.     

Identification of genes that function in the biogenesis and localization of small nucleolar RNAs in Saccharomyces cerevisiae

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.     

Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries

Applied Microbiology and Biotechnology - The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic...     

Identification of genes that influence gurken expression

Axial patterning in Drosophila relies on the deployment of patterning proteins at specific regions within the developing oocyte. This process involves transport of mRNAs from the nurse cells to the oocyte, localization of mRNAs within the oocyte, and translational regulation of these mRNAs to restrict the final distribution of the proteins. Despite extensive analysis, the events of deployment are not fully understood and it seems certain that many contributing factors remain to be identified. We describe the development and application of a sensitized genetic screen to reveal such additional factors. Overexpression of Imp, a factor implicated in regulation of gurken mRNA, causes a weak dorsalization that can be enhanced by reducing the level of other factors acting in the same pathway. A collection of deficiency mutants was screened using this assay, leading to the identification of 5 genes that are candidates to contribute to axial patterning. Three of the genes were characterized in greater detail. The mushroom body expressed gene was implicated in axial patterning, with overexpression leading to a range of patterning abnormalities that can be explained by a more primary defect in organization of the cytoskeleton. Two mitotic cell cycle control factors, cyclin E and E2f1, were also implicated, raising the possibility that a mitotic cell cycle checkpoint may impinge on grk expression, much as meiotic checkpoints can alter expression of this gene.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.

Principal Findings

Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.

Conclusions

We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.     

Identification of the last unknown genes in the fermentation pathway of lysine

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.     

Analysis of metabolic pathway using hybrid properties

Given a compounds-forming system, i.e., a system consisting of some compounds and their relationship, can it form a biologically meaningful pathway? It is a fundamental problem in systems biology. Nowadays, a lot of information on different organisms, at both genetic and metabolic levels, has been collected and stored in some specific databases. Based on these data, it is feasible to address such an essential problem. Metabolic pathway is one kind of compounds-forming systems and we analyzed them in yeast by extracting different (biological and graphic) features from each of the 13,736 compounds-forming systems, of which 136 are positive pathways, i.e., known metabolic pathway from KEGG; while 13,600 were negative. Each of these compounds-forming systems was represented by 144 features, of which 88 are graph features and 56 biological features. "Minimum Redundancy Maximum Relevance" and "Incremental Feature Selection" were utilized to analyze these features and 16 optimal features were selected as being able to predict a query compounds- forming system most successfully. It was found through Jackknife cross-validation that the overall success rate of identifying the positive pathways was 74.26%. It is anticipated that this novel approach and encouraging result may give meaningful illumination to investigate this important topic.     

Identification of genes by hybrid ribozymes that couple cleavage activity with the unwinding activity of an endogenous RNA helicase

Novel ribozymes that couple the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicase eIF4AI were constructed. This leads to extremely efficient cleavage of the target mRNA, regardless of the secondary structure of the RNA, and eliminates one of the major problems: many target sites on the RNA were previously inaccessible to cleavage due to secondary and/or tertiary structure formation. Moreover, libraries of hybrid ribozymes with randomized binding arms were introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype. Specifically, four genes known to be in the Fas-mediated apoptosis pathway were identified along with additional genes. This application of a randomized library of hybrid ribozymes represents a simple, powerful method for the identification of genes associated with specific phenotypes in the post-genome era.     

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation.     

Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.     

Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a genomic approach

Transforming growth factor (TGF)-beta-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-beta inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-beta-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-beta/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-beta regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-beta activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-beta induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-beta-induced apoptosis in RIE-1/Smad3 cells.