Sangdun Choi

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25–30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

A Bioinformatics Analysis of Memory Consolidation Reveals Involvement of the Transcription Factor c-Rel

Consolidation of long-term memory (LTM) is a complex process requiring synthesis of new mRNAs and proteins. Many studies have characterized the requirement for de novo mRNA and protein synthesis; however, few studies have comprehensively identified genes regulated during LTM consolidation. We show that consolidation of long-term contextual memory in the hippocampus triggers altered expression of numerous genes encompassing many aspects of neuronal function. Like contextual memory formation, this altered gene expression required NMDA receptor activation and was specific for situations in which the animal formed an association between a physical context and a sensory stimulus. Using a bioinformatics approach, we found that regulatory elements for several transcription factors are over-represented in the upstream region of genes regulated during consolidation of LTM. Using a knock-out mouse, we found that c-rel, one of the transcription factors identified in our bioinformatics study, is necessary for hippocampus-dependent long-term memory formation.

Overview of the Alliance for Cellular Signaling

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Enhanced T Cell Proliferation in Mice Lacking the p85β Subunit of Phosphoinositide 3-Kinase

Phosphoinositide 3-kinase activation is important for lymphocyte proliferation and survival. Disrupting the gene that encodes the major phosphoinositide 3-kinase regulatory isoform p85α impairs B cell development and proliferation. However, T cell functions are intact in the absence of p85α. In this study, we test the hypothesis that the related isoform p85β is an essential regulatory subunit for T cell signaling. Unexpectedly, T cells lacking p85β showed a marked increase in proliferation and decreased death when stimulated with anti-CD3 plus IL-2. Both CD4+ and CD8+ T cells completed more cell divisions. Transcriptional profiling revealed reduced levels of caspase-6 mRNA in p85β-deficient T cells, which was paralleled by reduced caspase-6 enzyme activity. Increased T cell accumulation was also observed in vivo following infection of p85β-deficient mice with mouse hepatitis virus. Together, these results suggest a unique role for p85β in limiting T cell expansion.

Analysis of the Major Patterns of B Cell Gene Expression Changes in Response to Short-Term Stimulation with 33 Single Ligands

We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca2+ and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.

Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation

BackgroundActivation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cl uster Assi gnment f or Biological I nference), which identifies significant co-clustering of genes with similar Gene Ontology™ annotation.ResultsCLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated.ConclusionThese observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation.

Gene expression profiles of light-induced apoptosis in arrestin/rhodopsin kinase-deficient mouse retinas

To examine the molecular processes that lead to light-induced retinal degeneration, mutant mice deficient in arrestin and rhodopsin kinase were raised in the dark and then subjected to relatively low doses of white light. The kinetics of the subsequent induction of apoptosis, change in mRNA transcript level, and photoreceptor cell death were monitored. Analysis of transcript profiles identified clusters of genes that responded differently to illumination, including a cluster of photoreceptor-specific genes that showed marked decreases in levels long before morphological damage could be readily ascertained. The behaviors of other gene clusters demonstrate the coordinate induction of stress gene responses early in the course of irradiation. There was little, if any, change in transcript levels corresponding to genes associated with the initiation of apoptosis or antiapoptotic effects. Transcript analysis provides insight into the patterns of gene expression that are associated with the different stages of retinal degeneration in this model system.

Dual ligand stimulation of RAW 264.7 cells uncovers feedback mechanisms that regulate TLR-mediated gene expression

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-γ, 2-methyl-thio-ATP (2MA), PGE2, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE2 and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-γ and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-γ-induced changes in gene expression at 1 h, IFN-γ signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-γ- response genes by augmenting STAT1 activity and by activating NF-κB, which synergizes with IFN-γ-induced transcriptional factors; 2) IFN-γ attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-β and inhibiting IL-10 gene expression; 4) PGE2 and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.

Enhancing RNAi with Synthetic RNA Duplexes

RNA interference (RNAi) is an evolutionarily conserved process by which specific mRNAs are targeted for degradation by complementary small interfering RNAs (siRNAs). It has become the method of choice for mammalian cell genetics and as a potential sequence-specific therapeutic approach (Sharp 1999; Hannon 2002; Hutvagner and Zamore 2002). Long double-stranded (ds) RNAs are degraded by the RNase III class endonuclease Dicer into 21- to 23-nt duplexes that have 2-base 3’-overhangs (Zamore et al. 2000; Bernstein et al. 2001). Dicer’s primary role in RNAi is the endonucleolytic processing of long dsRNAs into short 21- to 23-mer effector molecules (siRNAs) (Bernstein et al. 2001; Ketting et al. 2001). Dicer is also involved in the early steps of RNA induced silencing complex (RISC) complex formation and may be required for entry of the siRNA into RISC (Lee et al. 2004; Pham et al. 2004). In Drosophila, the protein R2D2 associates with Dicer (specifically Dicer-2) and binds siRNAs prior to entry into RISC (Liu et al. 2003). Although no functional homologue for R2D2 has been identified in mammals, a similar link is assumed to exist between the Dicer cleavage step and entry into RISC in mammalian cells. Human Dicer has been cloned and characterized (Zhang et al. 2002). Interestingly, while the recombinant enzyme can be used to process long dsRNA into functional siRNAs, the process is slow and complete “dicing” of a substrate in vitro can take 24h incubation (Zhang et al. 2002). Thus, other factors must be involved in promoting rapid Dicer cleavage in vivo.