Silencing of microRNAs in vivo with 'antagomirs'

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.     

A viral microRNA functions as an orthologue of cellular miR-155

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.     

Dicer recognizes the 5' end of RNA for efficient and accurate processing

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (～22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.     

The impact of microRNAs on protein output

MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.     

Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs （miRNAs） participate in the radiation-induced DNA damage response. However, a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thor- oughly assessed. We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation （IR）, quantified the ex- pression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms, Gene Ontol- ogy （GO） analysis, pathway analysis, and gene network construction. The results showed that the altered miRNAs were involved in regulation of various cellular functions, miRNA-gene network analyses revealed that miR- 186, miR- 106b, miR- 15 a/b, CCND 1 and CDK6 played vital role in the cellular radiation response. Using qRT-PCR, we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression. This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.     

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).     

Interferon modulation of cellular microRNAs as an antiviral mechanism

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.     

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.     

LNA-mediated microRNA silencing in non-human primates

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.