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Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

Background: MicroRNAs (miRNAs) are endogenous 21 to 23-nucleotide RNA molecules that regulate protein-coding gene expression in plants and animals via the RNA interference pathway. Hundreds of them have been identified in the last five years and very recent works indicate that their total number is still larger. Therefore miRNAs gene discovery remains an important aspect of understanding this new and still widely unknown regulation mechanism. Bioinformatics approaches have proved to be very useful toward this goal by guiding the experimental investigations., Results: In this work we describe our computational method for miRNA prediction and the results of its application to the discovery of novel mammalian miRNAs. We focus on genomic regions around already known miRNAs, in order to exploit the property that miRNAs are occasionally found in clusters. Starting with the known human, mouse and rat miRNAs we analyze 20 kb of flanking genomic regions for the presence of putative precursor miRNAs (pre-miRNAs). Each genome is analyzed separately, allowing us to study the species-specific identity and genome organization of miRNA loci. We only use cross-species comparisons to make conservative estimates of the number of novel miRNAs. Our ab initio method predicts between fifty and hundred novel pre-miRNAs for each of the considered species. Around 30% of these already have experimental support in a large set of cloned mammalian small RNAs. The validation rate among predicted cases that are conserved in at least one other species is higher, about 60%, and many of them have not been detected by prediction methods that used cross-species comparisons. A large fraction of the experimentally confirmed predictions correspond to an imprinted locus residing on chromosome 14 in human, 12 in mouse and 6 in rat. Our computational tool can be accessed on the world-wide-web., Conclusion: Our results show that the assumption that many miRNAs occur in clusters is fruitful for the discovery of novel miRNAs. Additionally we show that although the overall miRNA content in the observed clusters is very similar across the three considered species, the internal organization of the clusters changes in evolution.

MicroRNAs (miRNAs) are approximately 22 nt-long non-coding RNA molecules, believed to play important roles in gene regulation. We present a comprehensive analysis of the conservation and clustering patterns of known miRNAs in human. We show that human miRNA gene clustering is significantly higher than expected at random. A total of 37% of the known human miRNA genes analyzed in this study appear in clusters of two or more with pairwise chromosomal distances of at most 3000 nt. Comparison of the miRNA sequences with their homologs in four other organisms reveals a typical conservation pattern, persistent throughout the clusters. Furthermore, we show enrichment in the typical conservation patterns and other miRNA-like properties in the vicinity of known miRNA genes, compared with random genomic regions. This may imply that additional, yet unknown, miRNAs reside in these regions, consistent with the current recognition that there are overlooked miRNAs. Indeed, by comparing our predictions with cloning results and with identified miRNA genes in other mammals, we corroborate the predictions of 18 additional human miRNA genes in the vicinity of the previously known ones. Our study raises the proportion of clustered human miRNAs that are <3000 nt apart to 42%. This suggests that the clustering of miRNA genes is higher than currently acknowledged, alluding to its evolutionary and functional implications.

Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the oxytocin and VP magnocellular neurons of the hypothalamo-neurohypophysial system, to identify genes associated with the magnocellular neuron's adaptation to this physiological condition. Using laser microdissection of the SON, T7-based linear amplification of its RNA, and a 35,319-element cDNA microarray, we compare gene expression profiles between SONs in normoosmolar (control), 1-desamino-[8-D-arginine]-VP-treated normoosmolar, and hypoosmolar rats. We found 4959 genes with statistically significant differences in expression between normosmolar control and the hypoosmolar SONs, with 1564 of these differing in expression by more than 2-fold. These genes serve a wide variety of functions, and most were up-regulated in gene expression in hypoosmolar compared with control SONs. Of these, 90 were preferentially expressed in the SON, and 44 coded for transcription-related factors, of which 15 genes were down-regulated and 29 genes were up-regulated in the hypoosmolar rat SONs. None of these transcription-related factor genes significantly changed in expression after sustained 1-desamino-[8-D-arginine]-VP-treatment alone, indicating that these changes were associated with the hypoosmolar state and not due solely to a decreased activity in the SON. Quantitative in situ hybridization histochemistry was selectively used to confirm and extend these microarray observations. These results indicate that the hypoosmolar state is accompanied by a global, but selective, increase in expression of a wide variety of regulatory genes in the SON.

Purpose: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) regulate aqueous outflow by actively releasing ligands that upon binding to Schlemm's canal endothelial cells (SCEs) increase transendothelial flow, thereby facilitating the egress of aqueous., Methods: We tested our hypothesis by (1) activating the TMEs in vitro using a laser procedure known to increase aqueous outflow in vivo; (2) demonstrating that lasered TMEs become activated at the genome-wide level and synthesize ligands; (3) ascertaining that media conditioned by laser-activated TMEs and ligands therein increase transendothelial flow when added to SCEs; and (4) determining that ligands identified as synthesized by TMEs increase permeability when added to SCEs., Results: We find that adding either media conditioned by lasered TMEs or ligands synthesized by TMEs to naïve control SCEs increases permeability. Adding media boiled, diluted, or conditioned by nonlasered TMEs abrogates these permeability effects. Media conditioned by either lasered TMEs or SCEs (TME-cm/SCE-cm), when added to untreated controls of each cell type, induce congruous gene expression and flow effects: TME-cm induces far more differentially expressed genes (829 in control TMEs and 1,120 in control SCEs) than does the SCE-cm (12 in control TMEs and 328 in control SCEs), and TME-cm also increases flow much more (more than 11-fold in control TMEs and more than fourfold in control SCEs) than does the SCE-cm (fivefold in control TMEs and twofold in control SCEs)., Conclusions: As postulated, the TMEs release factors that regulate SCE permeability. Derangement of this TME-driven process may play an important role in the pathogenesis of glaucoma. Ligands identified, which regulate permeability, have potential use for glaucoma therapy.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).

Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins/epithelial sodium channel (DEG/ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1-0.5%), in the hot-plate test (52.5 and 55 vs. 50 degrees C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate- to high-intensity pain sensation.