Compilation of small RNA sequences, 1990.
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Animals↗
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Surprising discovery with a small RNA.
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Molecular Weight↗
Virus-host interactions: new insights from the small RNA world.
RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions.
Animals↗
Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding.
To quantitatively understand the binding affinity and target selectivity of small-molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub- optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.
Aminoglycosides↗
The small RNA profile during Drosophila melanogaster development.
Small RNAs ranging in size between 20 and 30 nucleotides are involved in different types of regulation of gene expression including mRNA degradation, translational repression, and chromatin modification. Here we describe the small RNA profile of Drosophila melanogaster as a function of development. We have cloned and sequenced over 4000 small RNAs, 560 of which have the characteristics of RNase III cleavage products. A nonredundant set of 62 miRNAs was identified. We also isolated 178 repeat-associated small interfering RNAs (rasiRNAs), which are cognate to transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs participate in defining chromatin structure. rasiRNAs are most abundant in testes and early embryos, where regulation of transposon activity is critical and dramatic changes in heterochromatin structure occur.
Animals↗
Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors.
RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.
Animals↗
Genetic and functional diversification of small RNA pathways in plants.
Multicellular eukaryotes produce small RNA molecules (approximately 21-24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.
Arabidopsis↗
The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae.
Quorum-sensing bacteria communicate with extracellular signal molecules called autoinducers. This process allows community-wide synchronization of gene expression. A screen for additional components of the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits revealed the protein Hfq. Hfq mediates interactions between small, regulatory RNAs (sRNAs) and specific messenger RNA (mRNA) targets. These interactions typically alter the stability of the target transcripts. We show that Hfq mediates the destabilization of the mRNA encoding the quorum-sensing master regulators LuxR (V. harveyi) and HapR (V. cholerae), implicating an sRNA in the circuit. Using a bioinformatics approach to identify putative sRNAs, we identified four candidate sRNAs in V. cholerae. The simultaneous deletion of all four sRNAs is required to stabilize hapR mRNA. We propose that Hfq, together with these sRNAs, creates an ultrasensitive regulatory switch that controls the critical transition into the high cell density, quorum-sensing mode.
Amino Acid Sequence↗
Small RNA viruses of insects: expression in plants and RNA silencing.
Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed.
Animals↗
CSRDB: a small RNA integrated database and browser resource for cereals.
Plant small RNAs (smRNAs), which include microRNAs (miRNAs), short interfering RNAs (siRNAs) and trans-acting siRNAs (ta-siRNAs), are emerging as significant components of epigenetic processes and of gene networks involved in development and in homeostasis. Here we present a bioinformatics resource for cereal crops, the Cereal Small RNA Database (CSRDB), consisting of large-scale datasets of maize and rice smRNA sequences generated by high-throughput pyrosequencing. The smRNA sequences have been mapped to the rice genome and to the available maize genome sequence and these results are presented in two genome browser datasets using the Generic Genome Browser. Potential RNA targets for the smRNAs have been predicted and access to the resulting smRNA/RNA target pair dataset has been made available through a MySQL based relational database. Various ways to access the data are provided including links from the genome browser to the target database. Data linking and integration are the main focus for this interface, and internal as well as external links are present. The resource is available at http://sundarlab.ucdavis.edu/smrnas/ and will be updated as more sequences become available.
Databases, Nucleic Acid↗
Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini.
When the cloned h22 histone gene repeat unit of the sea urchin Psammechinus miliaris is injected into the frog oocyte nucleus, transcription initiates faithfully in the promoter region of the H3 gene, but transcription termination or RNA processing to yield genuine H3 mRNA 3' ends is very inefficient. The generation of 3' ends can be enhanced by the injection of a small sea urchin poly (A)- RNA into the oocyte cytoplasm one day prior to injection of the histone gene repeat unit. This RNA is approximately 60 nucleotides in length and appears to be present in relatively low abundance in embryos of both Psammechinus and Paracentrotus. RNA extracted from a nuclear DEAE fraction, previously shown to complement the lesion in H3 gene expression, also causes the appearance of true 3' ends of H3 mRNA. Furthermore, RNA extracted from our 12S "termination" factor includes an RNA species comigrating with the 60 nucleotide RNA from poly (A)- RNA. We suggest that the 12S nuclear "termination" component is in fact a small nuclear RNP.
Animals↗
Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68.
A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers. The double-stranded form was cloned into the bacteriophage M1 3mp7. Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique. The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3. It also contained sequences homologous to segment 1.
Base Sequence↗
Regulation of RpoS by a novel small RNA: the characterization of RprA.
Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double-stranded RNA stem-loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double-strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem-loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non-essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.
Bacterial Proteins↗
Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.
Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.
Bacterial Proteins↗
Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.
Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.
Fluorescent Dyes↗
Invertebrate miRNA pva-small RNA-11881/pva-miR-11881 as a potential RNA-based therapeutic against white spot syndrome virus in infected shrimp.
Small RNAs and microRNAs (miRNAs) play diverse roles in host virus interactions and hold promise for therapeutic applications. An uncharacterized shrimp miRNA with potent activity against white spot syndrome virus (WSSV), a major double-stranded DNA pathogen in aquaculture, was identified and characterized. Among the 1,239 differentially expressed unannotated small RNAs in Penaeus vannamei hemocytes, one of the most strongly downregulated candidates, termed pva-small RNA-11881 or pva-miR-11881, was predicted to target multiple WSSV genes. A pva-small RNA-11881/pva-miR-11881 isomir that originates from the 5' untranslated region of a host lipase 3-like gene was identified. Its primary transcript contains Drosha and Dicer processing sites, and the precursor exhibits canonical pre-miRNA features. In vivo administration of its primary transcript, pva-pri-miR-11881, significantly reduced WSSV copy number and improved shrimp survival. Mechanistically, pva-miR-11881 directly suppresses crucial WSSV genes WSSV004, WSSV164, and WSSV419 and modulates the host immune response against WSSV infection by enhancing phenoloxidase activity, thereby reducing apoptosis and necrosis, and promoting caspase-1-mediated cell death. These findings reveal that the pva-miR-11881 in P. vannamei holds strong potential as a biotherapeutic agent for managing viral diseases in shrimp.
Animals↗
Chronological and geographical variations in the small RNA segment of the teratogenic Akabane virus.
Characterization of the small RNA segment of the teratogenic Akabane virus was performed to define the genetic heterogeneity and molecular epidemiology of the virus. The nucleotide and amino acid sequences were highly conserved among the Akabane virus isolates, irrespective of the year of isolation and geographical origin, except for a Kenyan strain MP496. On the basis of the nucleocapsid gene sequences of the segment, 170 field isolates were grouped into four distinct phylogenetic lineages. The Japanese isolates were divided into two major clusters, one containing the prototype strain JaGAr39 and strains isolated in the 1970s, 1988-1989, 1991, and 1994-1999, and the other containing strains isolated from 1982 to 1987, 1990, 1993, 1998, and 2000 to 2003 together with the Taiwanese and Israeli strains. The Asian strains seemed to have evolved in a common gene pool. The Australian and Kenyan strains were placed in the independent third and fourth clusters of the phylogenetic tree, respectively. No consecutive mutations have occurred on the small RNA segment of the Japanese isolates, indicating that the various genotypes of the virus might invade continuously from an exotic source and that certain viruses adaptable to the Japanese environment might spread transiently.
Animals↗
Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.
Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown.
Animals↗