Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.

In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myctransgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.T he bromodomain and extraterminal (BET) domain family of proteins Brd2, Brd3, Brd4, and BrdT bind via their tandem bromodomains (BD1 and BD2) to acetylated lysines in histones and other proteins (1). On binding, they regulate the transcription of genes critical for cell-cycle progression and apoptosis. Therefore, BET proteins have emerged as interesting proteins for targeted intervention of cancer.Recently, the small-molecule BET inhibitor (+)-JQ-1 (hereafter JQ1) was found to be a potent and specific suppressor of B cell-lineage malignancies (2, 3). In acute myelogenous leukemia, BRD4 is essential for tumor maintenance, and JQ1 recapitulates the effects of RNA interference of BRD4 (4, 5). JQ1 was subsequently shown to have an antiproliferative effect in other hematological malignancies and solid organ tumors including glioblastoma, prostate cancer, and neuroblastoma (6-10). The current model of how BET inhibitors (BETi) inhibit tumor cell proliferation places inhibition of MYC as mediating activity in lymphoid tumors, with Myc-independent activity in some solid tumor types such as lung adenocarcinoma (11). However, it has not been clear in hematopoietic tumor types whether the antiproliferative effects of BETi are mediated by suppression of MYC expression or whether effects on MYC are a correlative bystander of the mechanism, perhaps useful as a biomarker but not necessarily mechanistic (12).We have assessed the effect of RVX2135, a novel and orally bioavailable selective inhibitor of Brd2, Brd3, Brd4, and BrdT, in in vitro and in vivo models of Myc-induced lymphoma. We find that the effects are mediated by broad transcriptional changes and that these are genetically and functionally linked to histone deacetylase inhibitors. Results RVX2135 Blocks Proliferation of Myc-Induced Mouse Lymphoma Cellsand Induces Caspase-Dependent Apoptosis. RVX2135 is a novel small-molecule BET bromodoma...

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

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Small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. Resistance to invasive genetic material is conferred by the incorporation of short DNA sequences derived from this material into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by a CRISPR-associated (Cas) RNA endonuclease generates the mature effector RNAs that target foreign nucleic acid for degradation. Here we describe functional studies of a Cas5d ortholog, and high-resolution structural studies of a second Cas5d family member, demonstrating that Cas5d is a sequence-specific RNA endonuclease that cleaves CRISPR repeats and is thus responsible for processing of precrRNA. Analysis of the structural homology of Cas5d with the previously characterized Cse3 protein allows us to model the interaction of Cas5d with its RNA substrate and conclude that it is a member of a larger family of CRISPR RNA endonucleases.

Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo-and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. Double-stranded RNAs (dsRNAs)2 in eukaryotes are subject to a variety of processing reactions, including cleavage by the RNase III family members Drosha and Dicer in the micro RNA and small interfering RNA gene-silencing pathways and editing by members of the ADAR (adenosone deaminase that acts on RNA) enzyme family (1, 2). This latter reaction involves the hydrolytic deamination of adenosine (A) to inosine (I) within the context of dsRNA. Editing events of this type have been demonstrated in both cellular and viral transcripts and have been shown to function in altering the coding properties of the edited RNAs. For example, the life cycle of the Hepatitis ␦ virus is regulated by an editing event in the anti-genome in which a UAG stop codon is converted to a UIG tryptophan codon (3). An A to I modification is involved in the functional regulation of a growing number of cellular factors. These include the tissue-specific editing of the serotonin 5-HT2C receptor, which results in a reduction in response to serotonin agonists (4). Transcripts for subunits of the neural-specific AMPA class of glutamate-gated (GluR) ion channels undergo A to I modification at two positions, the Q/R and R/G editing sites, that affect the properties of the resulting channels (5, 6). In addition to the editing of these and other neuronal transcripts to effect codon changes, one deaminase family member, ADAR2, has been shown to autoregulate its own expression by the creation of a 3Ј-splice site (CAA to CAI) (7). Despite the identification of these editing substrates, the global role of A to I modification in higher eukaryotes remains unclear. Measurement of inosine levels in RNA isolated from rat tissue suggests a greater level of editing than indicated by known RNA substrates (8). A cloning protocol that depended upon an inosine-specific cleavage of RNA detected a large number of editing sites in non-coding regions of RNAs from Caenorhabditis elegans and humans that included sites in 5Ј-and 3Ј-untranslated regions and introns (9). Recent bioinformatic studies have suggested the presence of more than 12,000 editing sites corresponding to non-coding regions of t...

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