ABSTRACT
microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg(-1) LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg(-1) LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.
Subject(s)
Chlorocebus aethiops/genetics , Gene Silencing , MicroRNAs/genetics , Oligonucleotides/genetics , Animals , Female , Mice , Mice, Inbred C57BL , Oligonucleotides/administration & dosage , Oligonucleotides/adverse effectsABSTRACT
In metazoans, the nuclear export of bulk mRNAs is mediated by the export receptor TAP, together with its binding partner p15. A number of viral mRNAs, including the unspliced and partially spliced mRNA species of the human immunodeficiency virus (HIV), however, use an alternative export route via the importin beta-related export receptor CRM1. This raises the question of whether a subset of cellular mRNAs might be exported by CRM1 as well. To identify such mRNAs, we performed a systematic screen in different cell lines, using representational difference analyses of cDNA (cDNA-RDA). In HeLa and Cl-4 cells no cellular transcripts could be identified as exported via CRM1. In contrast, we found a number of CRM1-dependent mRNAs in Jurkat T cells, most of which are induced during a T cell response. One of the identified gene products, the dendritic cell marker CD83, was analyzed in detail. CD83 expression depends on a functional CRM1 pathway in activated Jurkat T cells as well as in a heterologous expression system, independent of activation. Our results point to an important role of the CRM1-dependent export pathway for the expression of CD83 and other genes under conditions of T cell activation.
Subject(s)
Karyopherins/metabolism , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , T-Lymphocytes/metabolism , Active Transport, Cell Nucleus , Antigens, CD/genetics , Base Sequence , Cell Line , DNA, Complementary/genetics , Gene Expression , Genes, env , HIV/genetics , HeLa Cells , Humans , Immunoglobulins/genetics , In Vitro Techniques , Jurkat Cells , Karyopherins/antagonists & inhibitors , Lymphocyte Activation/genetics , Membrane Glycoproteins/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , T-Lymphocytes/immunology , Exportin 1 Protein , CD83 AntigenABSTRACT
Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.
Subject(s)
Cytoplasmic Granules/metabolism , Gene Expression Regulation, Viral , Hepacivirus/physiology , RNA, Viral/metabolism , Virus Release/physiology , Cell Line, Tumor , Hepacivirus/genetics , Hepacivirus/metabolism , Hepacivirus/pathogenicity , Hepatocytes/virology , Humans , RNA, Viral/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Virus AssemblyABSTRACT
Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.
Subject(s)
Carrier Proteins/metabolism , Eukaryotic Initiation Factor-2/metabolism , Nuclear Proteins/metabolism , Oxidative Stress , Animals , Apoptosis , Arsenites/pharmacology , Carrier Proteins/genetics , DNA Helicases , Enzyme Inhibitors/pharmacology , Eukaryotic Initiation Factor-2/genetics , HeLa Cells/drug effects , Humans , Phosphorylation , Poly-ADP-Ribose Binding Proteins , Protein Biosynthesis/drug effects , RNA Helicases , RNA Recognition Motif Proteins , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Teratogens/pharmacology , Thapsigargin/pharmacology , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolismABSTRACT
MicroRNAs usually interact with 3' noncoding regions (3'NCRs) of target mRNAs leading to downregulation of mRNA expression. In contrast, liver-specific microRNA miR-122 interacts with the 5' end of the hepatitis C virus RNA genome, resulting in increased viral RNA abundance. We find that inserting the viral miR-122 binding site into the 3' noncoding region of a reporter mRNA leads to downregulation of mRNA expression, indicating that the location of the miR-122 binding site dictates its effect on gene regulation. Furthermore, we discovered an adjacent, second miR-122 binding site, separated from the first by a highly conserved 14-nucleotide sequence. Mutational analysis demonstrates that both miR-122 binding sites in a single viral genome are occupied by the microRNA and function cooperatively to regulate target gene expression. These findings set a paradigm for dual, position-dependent functions of tandem microRNA-binding sites. Targeting an oligomeric microRNA complex offers potential as an antiviral-intervention strategy.
Subject(s)
Genome, Viral , Hepacivirus/genetics , MicroRNAs/metabolism , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , 3' Untranslated Regions , 5' Untranslated Regions , Binding Sites , DNA Mutational Analysis , Genes, Reporter , RNA, Messenger/geneticsABSTRACT
Cellular responses to counter virus infection lead to the induction of cytoplasmic stress granules, which are composed of translationally stalled mRNAs. In this issue of Cell Host & Microbe, White and colleagues elucidate a mechanism where a poliovirus protease specifically cleaves a host cell factor involved in assembly of stress granules. This strategy ensures viral access to the limiting amounts of translation factors and interferes with host cell mRNA sorting.
Subject(s)
Cytoplasmic Granules/physiology , Viral Interference , Virus Diseases/immunology , Virus Diseases/virology , Virus Physiological Phenomena , Humans , Protein Biosynthesis , RNA Transport , RNA, Messenger/metabolism , Viral Proteins/metabolismABSTRACT
It has been known for some time that plants and insects use RNA interference (RNAi) as nucleic acid-based immunity against viral infections. However, it was unknown whether mammalian cells employ the RNA interference pathway as an antiviral mechanism as well. Over the past years, it has become clear that a variety of viruses, first in plants but recently in insect and mammalian viruses as well, encode suppressors of the RNAi pathway arguing for an antiviral role of this machinery. More recent findings have revealed that certain viruses encode their own microRNAs or microRNA-like RNA molecules, which are processed by the mammalian RNAi machinery. Furthermore, host-encoded microRNAs have been shown to both silence and enhance intracellular levels of viral RNAs. These findings argue that interactions between the RNAi pathway and viral genomes can profoundly affect the outcomes of the viral life cycles and contribute to the pathogenic signatures of the infectious agents.
Subject(s)
RNA Interference , Virus Diseases/virology , Virus Physiological Phenomena , Viruses/genetics , Animals , Mammals/genetics , Mammals/virology , MicroRNAs/metabolism , RNA/genetics , Signal Transduction/genetics , Virus Diseases/immunology , Virus Diseases/metabolism , Virus Replication/genetics , eIF-2 Kinase/metabolismABSTRACT
MicroRNAs (miRNAs), which can be expressed in a cell-type and tissue-specific manner, can influence the activities of genes that control cell growth and differentiation. Viruses often have clear tissue tropisms, raising the possibility that cellular miRNAs might modulate their pathogenesis. In this Review, we discuss recent findings that some vertebrate viruses either encode miRNAs or subvert cellular miRNAs, and that these miRNAs participate in both the infectious and the latent phase of the viral life cycle.
Subject(s)
DNA Viruses/genetics , DNA Viruses/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Animals , DNA Viruses/growth & development , Down-Regulation , Genome, Viral , Humans , Up-Regulation , Vertebrates/virologyABSTRACT
Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A(0), A(1), and A(2). Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.