Showing papers on "RNA published in 2007"
TL;DR: It is shown that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location, and it is proposed that this RNA is called “exosomal shuttle RNA” (esRNA).
Abstract: Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).
10,484 citations
Rockefeller University1, University of Basel2, Memorial Sloan Kettering Cancer Center3, Swiss Institute of Bioinformatics4, Sapienza University of Rome5, German Cancer Research Center6, Ludwig Maximilian University of Munich7, University of Freiburg8, Miltenyi Biotec9, J. Craig Venter Institute10, Columbia University11, University of Naples Federico II12, University of Düsseldorf13, University of Bonn14, Semmelweis University15, Yeshiva University16, National Institutes of Health17, Cornell University18
TL;DR: 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.
3,687 citations
TL;DR: Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes and support a highly interleaved organization of the human transcriptome.
Abstract: Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.
2,312 citations
TL;DR: Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics and coined to define this mechanism of inhibition of miRNA activity.
Abstract: MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.
1,767 citations
TL;DR: The proteins required for viRNA production as well as several key downstream components of the antiviral immunity pathway have been identified in plants, flies, and worms, illuminating an ongoing molecular arms race that likely impacts the evolution of both viral and host genomes.
1,431 citations
TL;DR: Several recently discovered examples of RNA operons in budding yeast, fruitfly and mammalian cells are described and their potential importance in processes such as immune response, oxidative metabolism, stress response, circadian rhythms and disease are described.
Abstract: Recent findings demonstrate that multiple mRNAs are co-regulated by one or more sequence-specific RNA-binding proteins that orchestrate their splicing, export, stability, localization and translation. These and other observations have given rise to a model in which mRNAs that encode functionally related proteins are coordinately regulated during cell growth and differentiation as post-transcriptional RNA operons or regulons, through a ribonucleoprotein-driven mechanism. Here I describe several recently discovered examples of RNA operons in budding yeast, fruitfly and mammalian cells, and their potential importance in processes such as immune response, oxidative metabolism, stress response, circadian rhythms and disease. I close by considering the evolutionary wiring and rewiring of these combinatorial post-transcriptional gene-expression networks.
1,261 citations
TL;DR: It is demonstrated that mammalian telomeres are transcribed into telomeric repeat–containing RNA (TERRA), and SMG factors represent a molecular link between TERRA regulation and the maintenance of telomere integrity.
Abstract: Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes, are essential for chromosome stability. Until now, telomeres have been considered to be transcriptionally silent. We demonstrate that mammalian telomeres are transcribed into telomeric repeat-containing RNA (TERRA). TERRA molecules are heterogeneous in length, are transcribed from several subtelomeric loci toward chromosome ends, and localize to telomeres. We also show that suppressors with morphogenetic defects in genitalia (SMG) proteins, which are effectors of nonsense-mediated messenger RNA decay, are enriched at telomeres in vivo, negatively regulate TERRA association with chromatin, and protect chromosome ends from telomere loss. Thus, telomeres are actively transcribed into TERRA, and SMG factors represent a molecular link between TERRA regulation and the maintenance of telomere integrity.
1,192 citations
TL;DR: A cytochrome P450 gene (CYP6AE14) is identified from cotton bollworm, which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol, and its expression correlates with larval growth when gOSSypol is included in the diet.
Abstract: We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
1,150 citations
TL;DR: RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells.
Abstract: Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species.
842 citations
TL;DR: In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level, and laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.
832 citations
TL;DR: In this article, a review focuses on recent advances, with emphasis on integration of small RNAs in stress regulatory networks, and shows that smallRNAs are involved in plant stress responses.
TL;DR: The fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity is proposed.
Abstract: The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity.
TL;DR: Repression of phosphoribosyl pyrophosphate synthetase 1, a target of the edited miR-376 RNA and an enzyme involved in the uric-acid synthesis pathway, contributes to tight and tissue-specific regulation of uri-acid levels, revealing a previously unknown role for RNA editing in miRNA-mediated gene silencing.
Abstract: Primary transcripts of certain microRNA (miRNA) genes are subject to RNA editing that converts adenosine to inosine. However, the importance of miRNA editing remains largely undetermined. Here we report that tissue-specific adenosine-to-inosine editing of miR-376 cluster transcripts leads to predominant expression of edited miR-376 isoform RNAs. One highly edited site is positioned in the middle of the 5'-proximal half "seed" region critical for the hybridization of miRNAs to targets. We provide evidence that the edited miR-376 RNA silences specifically a different set of genes. Repression of phosphoribosyl pyrophosphate synthetase 1, a target of the edited miR-376 RNA and an enzyme involved in the uric-acid synthesis pathway, contributes to tight and tissue-specific regulation of uric-acid levels, revealing a previously unknown role for RNA editing in miRNA-mediated gene silencing.
TL;DR: The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other nc RNPs.
Abstract: Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.
TL;DR: The transport of RNA molecules from the nucleus to the cytoplasm is fundamental for gene expression and understanding the mechanisms that connect RNP formation with export is a major challenge in the field.
Abstract: The transport of RNA molecules from the nucleus to the cytoplasm is fundamental for gene expression. The different RNA species that are produced in the nucleus are exported through the nuclear pore complexes via mobile export receptors. Small RNAs (such as tRNAs and microRNAs) follow relatively simple export routes by binding directly to export receptors. Large RNAs (such as ribosomal RNAs and mRNAs) assemble into complicated ribonucleoprotein (RNP) particles and recruit their exporters via class-specific adaptor proteins. Export of mRNAs is unique as it is extensively coupled to transcription (in yeast) and splicing (in metazoa). Understanding the mechanisms that connect RNP formation with export is a major challenge in the field.
TL;DR: RIG-I is a cytoplasmic sensor of HCV and is governed by RD interactions that are shared with LGP2 as an on/off switch controlling innate defenses, which may have therapeutic implications for immune regulation.
Abstract: RIG-I is an RNA helicase containing caspase activation and recruitment domains (CARDs). RNA binding and signaling by RIG-I are implicated in pathogen recognition and triggering of IFN-alpha/beta immune defenses that impact cell permissiveness for hepatitis C virus (HCV). Here we evaluated the processes that control RIG-I signaling. RNA binding studies and analysis of cells lacking RIG-I, or the related MDA5 protein, demonstrated that RIG-I, but not MDA5, efficiently binds to secondary structured HCV RNA to confer induction of IFN-beta expression. We also found that LGP2, a helicase related to RIG-I and MDA5 but lacking CARDs and functioning as a negative regulator of host defense, binds HCV RNA. In resting cells, RIG-I is maintained as a monomer in an autoinhibited state, but during virus infection and RNA binding it undergoes a conformation shift that promotes self-association and CARD interactions with the IPS-1 adaptor protein to signal IFN regulatory factor 3- and NF-kappaB-responsive genes. This reaction is governed by an internal repressor domain (RD) that controls RIG-I multimerization and IPS-1 interaction. Deletion of the RIG-I RD resulted in constitutive signaling to the IFN-beta promoter, whereas RD expression alone prevented signaling and increased cellular permissiveness to HCV. We identified an analogous RD within LGP2 that interacts in trans with RIG-I to ablate self-association and signaling. Thus, RIG-I is a cytoplasmic sensor of HCV and is governed by RD interactions that are shared with LGP2 as an on/off switch controlling innate defenses. Modulation of RIG-I/LGP2 interaction dynamics may have therapeutic implications for immune regulation.
TL;DR: In quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference.
Abstract: In recent years it has become clear that non-coding RNAs (that is, RNAs that are not translated into proteins) play a vital role in regulating gene expression. A previously unknown manifestation of that control has been found to operate on the human dihydrofolate reductase (DHFR) gene. The interfering RNA forms a complex with the DHFR gene's promoter region, and in so doing it interferes with the binding of transcription factors. The non-coding RNA is produced only in quiescent cells, leading to repression of the DHFR gene in these conditions. The DHFR major promoter is repressed in quiescent cells by a non-coding RNA initiated from an upstream promoter. This RNA molecule forms a complex with the major promoter, interacts with the general transcription factor IIB, and dissociates the pre-initiation complex from the promoter. Alternative promoters within the same gene are a general phenomenon in gene expression1,2. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.
TL;DR: The specificity of RNA silencing is conferred by small RNA guides that are processed from structured RNA or dsRNA, resulting in diversified pathways that control expression of endogenous and exogenous genes, invasive elements and viruses, and repeated sequences.
Abstract: The specificity of RNA silencing is conferred by small RNA guides that are processed from structured RNA or dsRNA. The core components for small RNA biogenesis and effector functions have proliferated and specialized in eukaryotic lineages, resulting in diversified pathways that control expression of endogenous and exogenous genes, invasive elements and viruses, and repeated sequences. Deployment of small RNA pathways for spatiotemporal regulation of the transcriptome has shaped the evolution of eukaryotic genomes and contributed to the complexity of multicellular organisms.
TL;DR: A highly conserved, thermodynamically stable RNA G-quadruplex is discovered in the 5' untranslated region (UTR) of the gene transcript of the human NRAS proto-oncogene and it is demonstrated that this NRAS RNAG- quadruplex modulates translation.
Abstract: Guanine-rich nucleic acid sequences can adopt noncanonical four-stranded secondary structures called guanine (G)-quadruplexes1. Bioinformatics analysis suggests that G-quadruplex motifs are prevalent in genomes2, which raises the need to elucidate their function. There is now evidence for the existence of DNA G-quadruplexes at telomeres with associated biological function3. A recent hypothesis supports the notion that gene promoter elements contain DNA G-quadruplex motifs that control gene expression at the transcriptional level4. We discovered a highly conserved, thermodynamically stable RNA G-quadruplex in the 5′ untranslated region (UTR) of the gene transcript of the human NRAS proto-oncogene. Using a cell-free translation system coupled to a reporter gene assay, we have demonstrated that this NRAS RNA G-quadruplex modulates translation. This is the first example of translational repression by an RNA G-quadruplex. Bioinformatics analysis has revealed 2,922 other 5′ UTR RNA G-quadruplex elements in the human genome. We propose that RNA G-quadruplexes in the 5′ UTR modulate gene expression at the translational level.
Abstract: The packaging of cytoplasmic mRNA into discrete RNA granules regulates gene expression by delaying the translation of specific transcripts. Specialized RNA granules found in germ cells direct the timing of maternal mRNA translation to promote germ cell development in the early embryo and establish the germ line for the next generation. Similarly, select neuronal mRNA transcripts are packaged into translationally inert RNA granules, transported to sites where their protein products are required, and only then activated and translated. Following translation, however, newly inactivated mRNAs released from polysomes can also be packaged into dynamic, transient structures known as stress granules (SGs) and processing bodies (PBs). Stress granules are composed largely of stalled preinitiation complexes, and contain mRNA, small ribosomal subunits, eIF3, eIF4E, eIF4G, and PABP, as their core components. PBs are associated with mRNA decay and contain the decapping enzymes DCP1/2, the 5' to 3' exonuclease Xrn1, the Lsm proteins (1-7), and the scaffolding proteins hedls/GE-1 and GW182. Both SGs and PBs contain mRNA, eIF4E, microRNAs and argonaute proteins, and various regulators of mRNA stability and translation (TTP, RCK/p54, and CPEB). Thus, SGs and PBs share some protein and mRNA components, but also contain a number of unique markers specific to each structure. We describe markers and staining procedures used to identify these distinct types of RNA granules, describe conditions that promote their assembly and disassembly, and establish YB-1 as a useful marker of SGs and PBs.
TL;DR: The sensors involved in coupling recognition of viruses to the induction of the type I IFN genes have only recently been uncovered and include endosomal and cytosolic receptors for RNA and DNA and their properties are reviewed.
TL;DR: Examination of micro-RNA abundance in the hippocampal region of fetal, adult and Alzheimer's disease brain indicates that micro-RNAs encoding miR-9, microR-124a,miR-125b, miM-128, miR -132 and miR –219 are abundantly represented in fetal hippocampus, are differentially regulated in aged brain, and an alteration in specific micro- RNA complexity occurs in Alzheimer hippocampus.
Abstract: Micro-RNAs constitute a family of small noncoding ribonucleic acids that are posttranscriptional regulators of messenger RNA activity. Although micro-RNAs are known to be dynamically regulated during neural development, the role of micro-RNAs in brain aging and neurodegeneration is not known. This study examined micro-RNA abundance in the hippocampal region of fetal, adult and Alzheimer's disease brain. The data indicate that micro-RNAs encoding miR-9, miR-124a, miR-125b, miR-128, miR-132 and miR-219 are abundantly represented in fetal hippocampus, are differentially regulated in aged brain, and an alteration in specific micro-RNA complexity occurs in Alzheimer hippocampus. These data are consistent with the idea that altered micro-RNA-mediated processing of messenger RNA populations may contribute to atypical mRNA abundance and neural dysfunction in Alzheimer's disease brain.
TL;DR: Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.
Abstract: RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.
TL;DR: H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-Tumor therapy.
Abstract: Background
Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target.
TL;DR: It is shown that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-β expression and that the signalling involves RIG-I, MDA5 and IPS-1.
Abstract: RNA molecules in virus-infected cells trigger interferon production and initiate antiviral innate immunity. Small RNA cleavage products from 'self' RNA generated by the antiviral endoribonuclease RNase L have now been found to initiate and amplify antiviral responses. This points to RNase L activation as a possible strategy for antiviral therapy. RNA molecules in virus-infected cells trigger interferon production and initiate antiviral innate immunity. This paper shows that small RNA cleavage products from 'self' RNA, generated by the antiviral endonuclease RNas L, can initiate and amplify antiviral responses. Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells1. These RNAs activate signalling cascades that activate the genes that encode α- and β-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs2,3,4,5. RIG-I and MDA5 interact with another CARD protein, interferon-β promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-κB to the IFN-β gene6,7,8,9,10. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L11, by 2′,5′-linked oligoadenylate (2-5A)12 produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-β expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3′ of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs13,14. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-β expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-β during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-β in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.
TL;DR: It is suggested that H19 expression results in the post-transcriptional downregulation of specific mRNAs during vertebrate development and can function as a primary microRNA precursor.
Abstract: Although H19 was the first imprinted noncoding transcript to be identified, the function of this conserved RNA has remained unclear. Here, we identify a 23-nucleotide microRNA derived from H19 that is endogenously expressed in human keratinocytes and neonatal mice and overexpressed in cells transfected with human or mouse H19 expression plasmids. These data demonstrate that H19 can function as a primary microRNA precursor and suggest that H19 expression results in the post-transcriptional downregulation of specific mRNAs during vertebrate development.
TL;DR: It is shown that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing, which underscores a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.
Abstract: hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a–mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.
TL;DR: It is found that transfection of expression constructs for MEG3 and its isoforms results in a significant increase in p53 protein levels and dramatically stimulates p 53-dependent transcription from a p53-responsive promoter, supporting the concept that M EG3 functions as a non-coding RNA.
TL;DR: In hepatoma cells that constitutively produce infectious HCV, HCV production is reduced by two agents that block VLDL assembly: an inhibitor of microsomal triglyceride transfer protein and siRNA directed against apoB.
Abstract: Hepatitis C virus (HCV) and triglyceride-rich very low-density lipoproteins (VLDLs) both are secreted uniquely by hepatocytes and circulate in blood in a complex. Here, we isolated from human hepatoma cells the membrane vesicles in which HCV replicates. These vesicles, which contain the HCV replication complex, are highly enriched in proteins required for VLDL assembly, including apolipoprotein B (apoB), apoE, and microsomal triglyceride transfer protein. In hepatoma cells that constitutively produce infectious HCV, HCV production is reduced by two agents that block VLDL assembly: an inhibitor of microsomal triglyceride transfer protein and siRNA directed against apoB. These results provide a possible explanation for the restriction of HCV production to the liver and suggest new cellular targets for treatment of HCV infection.
TL;DR: The results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples, and different formalin fixation times did not change the stability of miRNA based on real-time qRT-PCR analysis.
Abstract: microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 39 UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using highthroughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mg of fresh frozen and 1–5 mg of FFPE samples (correlation coefficient R 2 = 0.86–0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.