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PubMed2005

The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment.

Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Furthermore we find that haspin, a member of a distinctive group of protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis. Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays progression through early mitosis. This work reveals a new kinase involved in composing the histone code and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function during mitosis and meiosis.

Amino Acid Sequence↗
PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗
PubMed2006

Delivery strategies for siRNA-mediated gene silencing.

RNA interference (RNAi) represents a promising new gene silencing technology for functional genomics and a potential therapeutic strategy for a variety of genetic diseases. RNAi involves the targeted post-transcriptional degradation of messenger RNA thereby inhibiting the synthesis of the desired protein. This effectively leads to silencing of gene expression. The effectors of this process are short interfering RNA (siRNA) duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytosol. However, due to the large molecular weight and negative charge of siRNA duplexes the effective cellular uptake and intracellular delivery appear to represent a major challenge for the widespread use of RNAi in vivo. This review summarises some of the main delivery strategies that have been attempted for the transfection of siRNA to cells in vitro and in vivo.

Administration, Intranasal↗
PubMed2005

A sphingolipid elicitor-inducible mitogen-activated protein kinase is regulated by the small GTPase OsRac1 and heterotrimeric G-protein in rice 1[w].

Mitogen-activated protein kinase (MAPK) cascades are activated in plants during responses to pathogens or to pathogen-derived elicitors and mediate intracellular stress responses. Here, we show that a rice (Oryza sativa) MAPK, OsMAPK6, was posttranslationally activated in a cell culture by a sphingolipid elicitor. Suppression of OsMAPK6 expression by RNA interference resulted in a strong reduction of pathogen-induced Phe ammonia-lyase mRNA, whereas the mRNA level of another rice MAPK, OsMAPK5a, was highly increased. Silencing of a small GTPase, OsRac1, by RNA interference or loss-of-function mutation (d1) of the heterotrimeric G-protein alpha-subunit gene resulted in a strong reduction of the OsMAPK6 protein levels and of kinase activation by a sphingolipid elicitor. Furthermore, coimmunoprecipitation experiments with OsRac1 and OsMAPK6 proteins showed that OsMAPK6 is closely associated with the active form of OsRac1, but not with inactive forms of OsRac1. These results indicate that these two G-proteins regulate an elicitor-inducible MAPK in rice at the protein level.

Amino Acid Sequence↗
PubMed2006

Targeting leukemic fusion proteins with small interfering RNAs: recent advances and therapeutic potentials.

RNA interference has become an indispensable research tool to study gene functions in a wide variety of organisms. Because of their high efficacy and specificity, RNA interference-based approaches may also translate into new therapeutic strategies to treat human diseases. In particular, oncogenes such as leukemic fusion proteins, which arise from chromosomal translocations, are promising targets for such gene silencing approaches, because they are exclusively expressed in precancerous and cancerous tissues, and because they are frequently indispensable for maintaining the malignant phenotype. This review summarizes recent developments in targeting leukemia-specific genes and discusses problems and approaches for possible clinical applications.

Animals↗
PubMed2006

Principles and effects of microRNA-mediated post-transcriptional gene regulation.

MicroRNAs (miRNAs) are abundant regulatory RNAs involved in the regulation of many key biological processes. Recent advances in understanding the mechanism of RNA interference and miRNA-mediated mechanisms shed light on major principals of the formation of the regulatory complex and provide models to explain how these small regulatory RNA species interfere with gene expression and how they influence the translational status of the transcriptome.

Animals↗
PubMed1989

Probing Drosophila gene function by antisense RNA.

The conventional technique for assigning a particular genetic function to a cloned transcription unit has relied on the rescue of the mutant phenotype by germ line transformation. An alternative approach is to mimic a mutant phenotype by the use of antisense RNA injections to produce phenocopies. This approach has been successfully used to identify genes involved in early pattern forming processes in the Drosophila embryo. At the time when antisense RNA is injected, the embryo develops as a syncytium composed of about 5000 nuclei which share a common cytoplasm. The gene interactions required to establish the body plan occur before cellularization at the blastoderm stage. Thus the nuclei and their exported transcripts are accessible to the injected antisense RNA. The antisense RNA interferes with the endogenous RNA by an as yet unidentified mechanism. The extent of interference is only partial and produces phenocopies with characteristics of weak mutant alleles. In our lab and others, this approach has been successfully used to identify several genes required for normal Drosophila pattern formation.

Animals↗
PubMed2004

Inhibition of EGFP expression by siRNA in EGFP-stably expressing Huh-7 cells.

The RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally, and causes the degradation of an mRNA containing the same sequence. In this present study, an alternative approach was used to in vitro synthesize enhanced green fluorescent protein (EGFP) specific short interfering RNA (siRNA) using T7 RNA polymerase, and a pEGFP-N1 transfected, human hepatoma cell line Huh-7 derived Huh-7-N cell clone was established. When introduced the siRNA into the EGFP expressing Huh-7-N cells, the EGFP specific siRNA was able to specifically inhibit the expression of EGFP in Huh-7-N. In comparison with that in wild-type Huh-7 or that in Huh-7 co-transfected with pEGFP-N1, the inhibition of EGFP specific siRNA in Huh-7-N cells is more significant and repeatable. It is concluded that a cell clone Huh-7-N, which stably expresses EGFP, has been established, and the in vitro synthesized EGFP siRNA can be used in silencing the EGFP gene expression. This Huh-7-N/EGFP specific siRNA system has been proved reliable and convenient, and can also be applied widely as control in other RNA interference studies.

Cell Line↗
PubMed2006

Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Amino Acid Sequence↗
PubMed2005

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5' and 3'ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5' terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.

Aquaporin 4↗
PubMed2006

Partners in dicing.

Research into the mechanism of RNA interference has seen immense progress over the past few years. Recent studies of the protein Dicer, a key enzyme in the process, have started to reveal how this single enzyme is targeted to different RNA-silencing pathways.

Animals↗
PubMed2006

Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells.

Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.

Adenine Phosphoribosyltransferase↗
PubMed2006

Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3'hExo/ERI-1 family of 3' to 5' exonucleases.

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.

Amino Acid Sequence↗
PubMed2006

Natural selection drives extremely rapid evolution in antiviral RNAi genes.

RNA interference (RNAi) is perhaps best known as a laboratory tool. However, RNAi-related pathways represent an antiviral component of innate immunity in both plants and animals. Since viruses can protect themselves by suppressing RNAi, interaction between RNA viruses and host RNAi may represent an ancient coevolutionary "arms race." This could lead to strong directional selection on RNAi genes, but to date their evolution has not been studied. By comparing DNA sequences from different species of Drosophila, we show that the rate of amino acid evolution is substantially elevated in genes related to antiviral RNAi function (Dcr2, R2D2, and Ago2). They are among the fastest evolving 3% of all Drosophila genes; they evolve significantly faster than other components of innate immunity and faster than paralogous genes that mediate "housekeeping" functions. Based on DNA polymorphism data from three species of Drosophila, McDonald-Kreitman tests showed that this rapid evolution is due to strong positive selection. Furthermore, Dcr2 and Ago2 display reduced genetic diversity, indicative of a recent selective sweep in both genes. Together, these data show rapid adaptive evolution of the antiviral RNAi pathway in Drosophila. This is a signature of host-pathogen arms races and implies that the ancient battle between RNA viruses and host antiviral RNAi genes is active and significant in shaping RNAi function.

Amino Acid Sequence↗
PubMed2006

Effective gene suppression using small interfering RNA in hard-to-transfect human T cells.

RNA interference (RNAi) is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established proof of principle of small interfering RNA (siRNA) silencing in hard-to-transfect human T cell lines and primary human CD4 T cells. We used public and in-house programs to design four siRNAs each for GFP, for our novel cellular gene HALP, and for their corresponding scrambled siRNA controls. We generated siRNA expression cassettes (SECs) by PCR and directly transfected the PCR products into T cells using amaxa Nucleofector technology. The most effective SECs were selected and cloned into a TA cloning vector and titered with their respective controls to increase transfection efficiency. Flow cytometry and fluorescence microscopy analyses were performed for GFP siRNAs, and Northern blot analysis was done to assess the HALP silencing effect. These experiments demonstrate that SECs are an excellent screening tool to identify siRNA sequences effective in silencing expression of genes of interest. The vector expressing the most effective siRNA robustly inhibited GFP expression (up to 92%) in the context of co-transfection in human T cell lines and primary CD4 T cells. The optimized siRNA for our endogenous cellular gene HALP also silenced its target RNA expression by more than 90%. These studies demonstrate that the combination of SEC, siRNA expression vectors and Nucleofector technology can be successfully applied to hard-to-transfect human T cell lines and primary T cells to effectively silence genes.

Asparaginase↗
PubMed2005

Modulation of angiogenesis with siRNA inhibitors for novel therapeutics.

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.

Cell Adhesion Molecules↗
PubMed2005

Detection of foreign RNA: implications for RNAi.

RNA interference (RNAi) is an exciting technology with applications in basic research, in elucidation and validation of drug targets, and as a direct therapeutic. In mammalian settings, it is based on the introduction or expression of small interfering RNA (siRNA) that guide the cleavage of a complementary target messenger RNA. While siRNA certainly directs specific silencing of genes in mammalian cells, longer RNA typically used to silence genes in other organisms potently activate mammalian cell defence mechanisms leading to a non-specific halt in translation, to activation of transcription and often, to cell death. Recent research has revealed that siRNA in certain settings can also activate these RNA-responsive pathways. With the recent advances in RNAi technology and its first forays into the in vivo setting now coming to light, it is pertinent to review the cellular response to ribonucleic acids typically used in RNAi methods.

Animals↗