Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.

Non-coding RNAs as drug targets.

Most of the human genome encodes RNAs that do not code for proteins. These non-coding RNAs (ncRNAs) may affect normal gene expression and disease progression, making them a new class of targets for drug discovery. Because their mechanisms of action are often novel, developing drugs to target ncRNAs will involve equally novel challenges. However, many potential problems may already have been solved during the development of technologies to target mRNA. Here, we discuss the growing field of ncRNA - including microRNA, intronic RNA, repetitive RNA and long non-coding RNA - and assess the potential and challenges in their therapeutic exploitation.

HP1BP3, a Chromatin Retention Factor for Co-transcriptional MicroRNA Processing.

Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.

Activating frataxin expression by repeat-targeted nucleic acids.

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

Argonaute 2-dependent Regulation of Gene Expression by Single-stranded miRNA Mimics.

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression. Aberrant expression of miRNAs can affect development of cancer and other diseases. Synthetic miRNA mimics can modulate gene expression and offer an approach to therapy. Inside cells, mature miRNAs are produced as double-stranded RNAs and miRNA mimics typically retain both strands. This need for two strands has the potential to complicate drug development. Recently, synthetic chemically modified single-stranded silencing RNAs (ss-siRNA) have been shown to function through the RNAi pathway to induce gene silencing in cell culture and animals. Here, we test the hypothesis that single-stranded miRNA (ss-miRNA) can also mimic the function of miRNAs. We show that ss-miRNAs can act as miRNA mimics to silence the expression of target genes. Gene silencing requires expression of argonaute 2 (AGO2) protein and involves recruitment of AGO2 to the target transcripts. Chemically modified ss-miRNAs function effectively inside cells through endogenous RNAi pathways and broaden the options for miRNA-based oligonucleotide drug development.

Reduced Expression of Argonaute 1, Argonaute 2, and TRBP Changes Levels and Intracellular Distribution of RNAi Factors.

Until recently, Argonaute 2 (AGO2) and other RNA factors were believed to be restricted to the cytoplasm of mammalian somatic cells. It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function. We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei. Inhibition of AGO1 expression led to increased AGO2 levels, while knockdown of AGO2 led to increased levels of AGO1. Blocking AGO1, AGO2, or TRBP expression changed expression levels and nuclear distribution of RNAi factors Dicer, TNRC6A (GW182), and TRBP. These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.

Effect of 2'-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells.

Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2'-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.

Promoter RNA links transcriptional regulation of inflammatory pathway genes.

Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.

Transcriptional silencing by single-stranded RNAs targeting a noncoding RNA that overlaps a gene promoter.

RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

Enhanced binding of trigonal DNA-carbohydrate conjugates to lectin.

Novel trigonal DNA-carbohydrate conjugates were prepared and evaluated to explore efficient carbohydrate-lectin interactions. Carbohydrate-modified oligonucleotides were enzymatically prepared, then hybridized to form 3-way junction DNAs. The thermal stabilities of the junctions were assessed by UV melting analysis and formation of constructs was confirmed by gel electrophoresis. Fluorescence titration assays revealed that the trigonal DNA-carbohydrate conjugates exhibit high affinity to lectins depending on the distribution of carbohydrates presented in each arm. These results suggest that self-assembled 3-way DNA architectures could offer a useful platform for controlling the spatial distribution of carbohydrates on conjugates and achieving more efficient molecular recognition.

Allele-selective inhibition of trinucleotide repeat genes.

Expanded trinucleotide repeats cause Huntington's disease (HD) and many other neurodegenerative disorders. There are no cures for these devastating illnesses and treatments are urgently needed. Each trinucleotide repeat disorder is the result of the mutation of just one gene, and agents that block expression of the mutant gene offer a promising option for treatment. Therapies that block expression of both mutant and wild-type alleles can have adverse effects, challenging researchers to develop strategies to lower levels of mutant protein while leaving adequate wild-type protein levels. Here, we review approaches that use synthetic nucleic acids to inhibit expression of trinucleotide repeat genes.

Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter.

Low-density lipoprotein receptor (LDLR) is a cell-surface receptor that plays a central role in regulating cholesterol levels. Increased levels of LDLR would lead to reduced cholesterol levels and contribute to strategies designed to treat hypercholesterolemia. We have previously shown that duplex RNAs complementary to transcription start sites can associate with noncoding transcripts and activate gene expression. Here we show that duplex RNAs complementary to the promoter of LDLR activate expression of LDLR and increase the display of LDLR on the surface of liver cells. Activation requires complementarity to the LDLR promoter and can be achieved by chemically modified duplex RNAs. Promoter-targeted duplex RNAs can overcome repression of LDLR expression by 25-hydroxycholesterol and do not interfere with activation of LDLR expression by lovastatin. These data demonstrate that small RNAs can activate LDLR expression and affect LDLR function.

Allele-selective inhibition of mutant huntingtin by peptide nucleic acid-peptide conjugates, locked nucleic acid, and small interfering RNA.

The ability to inhibit expression of a mutant allele while retaining expression of a wild-type protein might provide a useful approach to treating Huntington's Disease (HD) and other inherited pathologies. The mutant form of huntingtin (HTT), the protein responsible for HD, is encoded by an mRNA containing an expanded CAG repeat. We demonstrate that peptide nucleic acid conjugates and locked nucleic acids complementary to the CAG repeat selectively block expression of mutant HTT. The selectivity of inhibition is at least as good as that shown by a small interfering RNA targeted to a deletion polymorphism. Our data suggest that antisense oligomers are promising subjects for further development as an anti-HD therapeutic strategy.

Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs.

Expanded trinucleotide repeats cause many neurological diseases. These include Machado-Joseph disease (MJD) and Huntington's disease (HD), which are caused by expanded CAG repeats within an allele of the ataxin-3 (ATXN3) and huntingtin (HTT) genes, respectively. Silencing expression of these genes is a promising therapeutic strategy, but indiscriminate inhibition of both the mutant and wild-type alleles may lead to toxicity, and allele-specific approaches have required polymorphisms that differ among individuals. We report that peptide nucleic acid and locked nucleic acid antisense oligomers that target CAG repeats can preferentially inhibit mutant ataxin-3 and HTT protein expression in cultured cells. Duplex RNAs were less selective than single-stranded oligomers. The activity of the peptide nucleic acids does not involve inhibition of transcription, and differences in mRNA secondary structure or the number of oligomer binding sites may be important. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length may offer new options for developing treatments for MJD, HD and related hereditary diseases.

[Ten cases of acute superior mesenteric artery occlusion].

This report deals with the perioperative management of ten patients with acute superior mesenteric artery occlusion. All ten patients survived after the surgery. Anesthesia in six patients were maintained with isoflurane, and in four patients with propofol, fentanyl and ketamine (PFK). Total intravenous anesthesia with PFK may provide stable anesthetic managements in superior mesenteric artery occlusion. After the operation patients were treated to control severe infection, multiple organ failure and coagulopathy. Three patients who have only 7, 10 or 5 cm of jejunum are undergoing home parenteral nutrition for short bowel syndrome. Early treatment of intestinal ischemia is essential because necrosis may develop as the condition progresses. The intestine should be preserved to the great extent possible in surgery for acute superior mesenteric arterial occlusion to obtain good long-term quality of life.

Construction of saccharide-modified DNAs by DNA polymerase.

Novel deoxyribonucleotide triphosphates bearing maltose or lactose groups were synthesized as substrates for DNA polymerase. The incorporation efficiencies of these modified substrates were investigated in both primer extension reactions and PCR. The stability and conformation of saccharide-modified dsDNAs were assessed by UV absorbance melting experiments and CD analysis. Enzymatic incorporation of saccharide-modified substrates can be used for the efficient production of saccharide-modified DNAs.

Ectopic varices in a right diaphragm that ruptured into the pleural cavity.

The term ectopic varices is used to describe dilated portosystemic collateral veins in unusual locations other than the gastroesophageal region. We recently experienced a rare case of ectopic varices that developed in the right diaphragm and ruptured into the pleural cavity. A 68-year-old female with hepatocellular carcinoma complicated with liver cirrhosis was admitted due to an acute onset of dyspnea and right bloody pleural effusion. Because of the patient's advanced hepatocellular carcinoma and poor condition, conservative therapies such as hemostats and blood transfusion were selected. Even though the bleeding to the pleural cavity stopped spontaneously, the patient died due to a progression of liver failure. Autopsy revealed a huge collateral vein in the right diaphragm. The etiology, prevalence, relationship with portal hypertension, and treatment of ectopic varices are discussed herein.

Enricher for fraction of high-pressure liquid chromatography.

A device is developed for concentrating a dilute solution without losing the components with boiling points slightly higher than the solvent. The device consists of an evaporator, receptor, and approximately 100 capillaries. A dilute solution is introduced into the evaporator and heated at a lower temperature than the boiling point of the solvent with the addition of a helium gas flow. As a result, mostly only the solvent evaporates, passes through the capillaries, and enters into the receptor. The low-boiling-point components in the solute, with boiling points slightly higher than the solvent, are trapped at the inlet of the capillaries. These components are then recovered by a small amount of solvent supplied from the receptor through the capillaries, with the main components of the solute concentrated at the bottom of the evaporator. A diesel fuel is separated into aliphatic and aromatic fractions by high-pressure liquid chromatography using a silica gel column. These fractions are then analyzed by low-resolution field ionization mass spectrometry, following concentration using the described device. The analytical results show that the final composition of the fractions is almost the same as that of the aliphatic or aromatic hydrocarbons in the original fuel.

Severe manifestation of acute hepatitis A recently found in Gunma, Japan.

BACKGROUND: The incidence of acute hepatitis A infection in Japan peaked 10 years ago and has been decreasing since then. However, an increase in severe cases of the disease has been documented recently. We experienced an outbreak in 1998-1999, and compared the clinical features of the disease in 1998-1999 (recent outbreak) and in 1987-1988 (past outbreak) in our prefecture (Gunma). METHODS: Forty patients with acute hepatitis A were admitted to nine Gunma hospitals from October 1998 to September 1999. Their clinical features were compared with those of 100 patients with acute hepatitis A admitted to the same hospitals in 1987-1988. RESULTS: Both outbreaks occurred mostly during the winter-spring season. Secondary familial infection was significantly decreased in the recent outbreak. Patients in the recent outbreak were 7 years older than those in the past outbreak. Laboratory findings, such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and prothrombin time, were worse in the recent than in the past outbreak. Severe-type hepatitis and fulminant hepatitis occurred in 5 patients (12.5%) in the recent outbreak but in only 2 patients (2.0%) in the past outbreak. CONCLUSIONS: Clinical data and manifestations were more severe in the recent outbreak than in the past outbreak of acute hepatitis A. It is important to be aware of hepatitis A virus infection and to take into account the available vaccination against hepatitis A virus in Japan.