The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Cellular levels of key regulatory proteins are controlled via ubiquitination and subsequent degradation. Deubiquitinating enzymes or isopeptidases can potentially prevent targeted destruction of protein substrates through deubiquitination prior to proteasomal degradation. However, only one deubiquitinating enzyme to date has been matched to a specific substrate in mammalian cells and shown to functionally modify it. Here we show that the isopeptidase USP2a (ubiquitin-specific protease-2a) interacts with and stabilizes fatty acid synthase (FAS), which is often overexpressed in biologically aggressive human tumors. Further, USP2a is androgen-regulated and overexpressed in prostate cancer, and its functional inactivation results in decreased FAS protein and enhanced apoptosis. Thus, the isopeptidase USP2a plays a critical role in prostate cancer cell survival through FAS stabilization and represents a therapeutic target in prostate cancer.

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs). Analysis of >2 million images, acquired by quantitative fluorescence microscopy, showed that depletion of 1,152 genes strongly affected cell-cycle progression. These genes clustered into eight distinct phenotypic categories based on phase of arrest, nuclear area, and nuclear morphology. Phase-specific networks were built by interrogating knowledge-based and physical interaction databases with identified genes. Genome-wide analysis of cell-cycle regulators revealed a number of kinase, phosphatase, and proteolytic proteins and also suggests that processes thought to regulate G 1-S phase progression like receptor-mediated signaling, nutrient status, and translation also play important roles in the regulation of G 2͞M phase transition. Moreover, 15 genes that are integral to TNF͞NF-B signaling were found to regulate G 2͞M, a previously unanticipated role for this pathway. These analyses provide systems-level insight into both known and novel genes as well as pathways that regulate cell-cycle progression, a number of which may provide new therapeutic approaches for the treatment of cancer.high-content screening ͉ network analysis ͉ small interfering RNA ͉ human genome

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A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity.

Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)–TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1␤ (IL-1␤) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1␤, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-B and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways. Tumor necrosis factor (TNF)2 is a pleiotropic mediator of a wide range of cellular responses to infection, such as cytokine and chemokine production, cell migration, cell death, and cell differentiation and maturation (1). TNF plays a pivotal role in several autoimmune disorders such as rheumatoid arthritis; this is underscored by the clinical success of neutralizing TNF with antibodies or soluble receptors (2). A better understanding of intracellular TNF signaling is therefore of high clinical relevance.The two TNF receptors, TNFR1 (p55, TNFRSF1A) and TNFR2 (p75, TNFRSF1B), show high homology in their extracellular domains but less in their intracellular domains. Although soluble TNF binds TNFR1 with higher affinity than TNFR2 and therefore acts primarily via TNFR1, membranebound TNF activates equally TNFR1 and TNFR2 (3). In most tissues TNF signaling is mediated by TNFR1, whereas TNFR2 is restricted to fewer specific tissues, mostly of an immunological nature (3). Upon ligand binding, TNFR1 trimerizes and recruits TNF receptor-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1), and TNF receptor-associated factor 2 (TRAF2). This first complex acts as a platform at the plasma membrane to activate the NF-B and MAPK signaling cascades, promoting cell survival and the expression of inflammatory cytokines. In a second step, TNFR1 is internalized into endocytic vesicles together with TRADD and RIP1 and recruits the proapoptotic molecules Fas-associated death domain (FADD) and caspase-8. This complex will initiate the apoptotic cell death program if concurrent anti-apoptotic NF-B activation is absent (4).The TGF␤-activated kinase 1 (TAK1) complex has a central function in many inflammatory pathways such as the TNFR, interleukin 1␤ receptor (IL-1R), and several Toll-...

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.