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PubMed2004

A systemic small RNA signaling system in plants.

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.

Amino Acid Sequence↗
PubMed2026

When light colour matters: Spectral quality is associated with distinct small RNA candidates in Arabidopsis thaliana.

The spectral composition of incident light is perceived by plant photoreceptors and can rapidly reshape downstream gene expression programs; however, its impact on the small-RNA layer beyond annotated miRNAs remains incompletely characterized. The objective of this study was to determine whether 3 h exposure of Arabidopsis thaliana rosettes to blue, green, red, or white light at an equal photosynthetic photon flux density (PPFD) of 400 μmol m-2 s-1 is associated with distinct profiles of candidate small RNAs. A stringent discovery and post-processing workflow was applied to identify high-confidence, treatment-associated small RNA candidates beyond annotated miRNA. The miRkwood-based pipeline, combined with additional filtering and contextual annotation, recovered a compact set of candidates dominated by the blue-light treatment (41 candidates), compared with fewer candidates associated with red (8), green (6), and white light (3). Genomic-context analysis indicated that most candidate sites were intergenic, with candidates detected under green light being entirely intergenic, and overlap with transposon annotations was used to distinguish candidates arising from transposon-rich genomic space. Sequence-feature profiling revealed pronounced treatment-dependent terminal nucleotide biases at both 5' and 3' ends, consistent with spectrum-associated shifts in length-class and terminal-nucleotide signatures that are informative for AGO-loading hypotheses. Target prediction highlighted a subset of genes showing convergent targeting by multiple independent blue-associated candidates, and duplex summaries showed structured, plant-like complementarity patterns (including frequent 10-11 pairing). Together, these results indicate that 3 h exposure to wavelength-defined light at 400 μmol m-2 s-1, particularly blue light, is associated with a distinct profile of detectable candidate small RNAs in Arabidopsis leaves and identify candidate interactions for follow-up validation.

5′ nucleotide bias↗
PubMed2005

Pathways through the small RNA world of plants.

RNA silencing pathways in plants have diversified along with key gene families involved in small RNA biogenesis and effector steps. Evidence suggests that these pathways have distinct roles in plant biology.

Amino Acid Sequence↗
PubMed2002

Detecting ligand binding to a small RNA target via saturation transfer difference NMR experiments in D(2)O and H(2)O.

A small RNA motif is used as a target for ligand-based NMR-screening by saturation transfer difference (STD) NMR experiments. The prerequisites for using a small RNA target in STD experiments, such as saturation time, frequency, and pulses, are discussed. We also show that it is of advantage to use D2O as solvent instead of H2O due to the reduced R1 relaxation rate in D2O. The 27-nucleotide model of the ribosomal A-site was known to bind the aminoglycoside paromomycin with high affinity. This binding interaction could be detected easily, proving the effectiveness of STD NMR experiments as a screening tool for RNA-ligand interactions.

Anti-Bacterial Agents↗
PubMed1990

Translational control by the Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates.

A role for the Epstein-Barr virus small RNA species EBER-1 in the regulation of protein synthesis has been investigated in the reticulocyte-lysate cell-free translation system. Recombinant EBER-1 was synthesized by in vitro transcription of a plasmid containing the viral gene and purified by CF11-cellulose chromatography and ribonuclease III treatment. When added to the reticulocyte lysate at 10-20 micrograms/ml or more, EBER-1 prevents the inhibition of protein synthesis caused by low concentrations of synthetic double-stranded RNA, poly(I).poly(C). This effect is eliminated by treatment of the recombinant EBER-1 with ribonuclease T1. Disruption of the secondary structure of EBER-1 by substitution of inosine for guanosine in the in-vitro-synthesized RNA impairs the ability of EBER-1 to prevent the poly(I).poly(C)-mediated inhibition of protein synthesis. These results suggest that high concentrations of EBER-1 regulate protein synthesis by blocking the activation of the double-stranded RNA-dependent eukaryotic initiation factor 2 alpha (eIF-2 alpha) protein kinase DAI (p68), and that this property is dependent on the secondary structure of the small RNA molecule.

Animals↗
PubMed2006

Exploring the energy landscape of a small RNA hairpin.

The energy landscape of a small RNA tetraloop hairpin is explored by temperature jump kinetics and base-substitution. The folding kinetics are single-exponential near the folding transition midpoint T(m). An additional fast phase appears below the midpoint, and an additional slow phase appears above the midpoint. Stem mutation affects the high-temperature phase, while loop mutation affects the low-temperature phase. An adjusted 2-D lattice model reproduces the temperature-dependent phases, although it oversimplifies the structural interpretation. A four-state free energy landscape model is generated based on the lattice model. This model explains the thermodynamics and multiphase kinetics over the full temperature range of the experiments. An analysis of three variants shows that one of the intermediate RNA structures is a stacking-related trap affected by stem but not loop modification, while the other is an early intermediate that forms some stem and loop structure. Even a very fast-folding 8-mer RNA with an ideal tetraloop sequence has a rugged energy landscape, ideal for testing analytical and computational models.

Kinetics↗
PubMed1987

Brain-specific small RNA during development and ageing of mice.

The ID sequence is a repetitive sequence family of rodent species that is transcribed specifically as small RNA in the brain. The amount of brain-specific small RNA in mice was shown to increase greatly in the first month after birth and then to remain unchanged until senescence. This RNA was suggested to be involved in regulation of brain-specific gene expression during postnatal development.

Aging↗
PubMed1999

Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA.

Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.

Amino Acid Sequence↗
PubMed1980

A small RNA induced late in simian virus 40 infection can associate with early viral mRNAs.

Analysis of total cytoplasmic and polyadenylylated cytoplasmic RNA from cells lytically infected with simian virus 40 (SV40) has demonstrated the presence of a small RNA, approximately 65 nucleotides long, that is induced late in lytic infection. This small RNA is apparently specific in size and sequence and is not selected on columns of oligo(dT)-cellulose. It is homologous to a region of the early SV40 mRNAs (and to the late DNA strand), starting approximately 250 nucleotides from the 3' end of the early mRNAs (SV40 map position 0.21). The function of this RNA in the viral cycle and its source are unknown at this time; however, its temporal expression, unique sequence, and interesting region of homology within the SV40 genome suggest a possible role in the control of SV40 gene expression.

Animals↗
PubMed2005

The developmental miRNA profiles of zebrafish as determined by small RNA cloning.

MicroRNAs (miRNAs) represent a family of small, regulatory, noncoding RNAs that are found in plants and animals. Here, we describe the miRNA profile of the zebrafish Danio rerio resolved in a developmental and cell-type-specific manner. The profiles were obtained from larger-scale sequencing of small RNA libraries prepared from developmentally staged zebrafish, and two adult fibroblast cell lines derived from the caudal fin (ZFL) and the liver epithelium (SJD). We identified a total of 154 distinct miRNAs expressed from 343 miRNA genes. Other experimental/computational sources support an additional 10 miRNAs encoded by 19 genes. The miRNAs can be classified into 87 distinct families. Cross-species comparison indicates that 81 families are conserved in mammals, 17 of which also have at least one member conserved in an invertebrate. Our analysis reveals that the zygotes are essentially devoid of miRNAs and that their expression begins during the blastula period with a zebrafish-specific family of miRNAs encoded by closely spaced multicopy genes. Computational predictions of zebrafish miRNA targets are provided that take into account the depth of evolutionary conservation. Besides miRNAs, we identified a prominent class of repeat-associated small interfering RNAs (rasiRNAs).

Animals↗
PubMed1999

Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA.

Small cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.

Bacillus subtilis↗
PubMed2005

A universal plasmid library encoding all permutations of small interfering RNA.

Small interfering RNA (siRNA) is normally designed to silence preselected known genes. Such selections are inevitably prone to bias as a result of limited knowledge about the biological process, transcript identity, and functions. A library that contains all permutations of siRNA could avoid such problems. In this paper, it is shown that 5 x 10(7) siRNA-encoding plasmids can be constructed in a single tube by using vectors with two mutated RNA polymerase III promoters arranged in a convergent manner. Such a library was used to carry out genomewide screening of functional genes in a phenotype-driven manner. Multiple siRNAs that induce a significant increase of cell proliferation speed were identified.

Base Sequence↗
PubMed2004

The RITS complex-A direct link between small RNA and heterochromatin.

In a recent report, Moazed, Grewal, and colleagues (Verdel et al., 2004) characterize the RITS (RNA induced initiation of transcriptional silencing) protein complex in fission yeast. They provide a sought-for link between the small RNA produced by the RNA interference machinery and heterochromatin components, suggesting a mechanism for how heterochromatin formation can be targeted in trans to specific chromosomal regions.

Fungal Proteins↗
PubMed2006

An antibody-based microarray assay for small RNA detection.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

Antibodies, Monoclonal↗
PubMed2004

Improved silencing vector co-expressing GFP and small hairpin RNA.

Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells. The GFP signal was a reliable reporter (93%-98%) of strong knockdown (approximately 90%) over a wide range of GFP intensities. The GFP reporter made possible the application of fluorescent-activated cell sorting (FACS) to purify the knockdown cell population. Such populations facilitated Western blotting analysis to determine depletion of the target protein. pG-SUPER was also applied to evaluate gene replacement by exogenous genes rendered refractory to siRNA by introducing silent mutations. Recovery of lamin A was linearly correlated to the expression level of the rescue gene. pG-SUPER will expand plasmid-based siRNA applications through the easy and reliable detection of knockdown and rescued cells.

Gene Silencing↗
PubMed1989

Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA.

A 13 kDa protein, covalently linked to a small RNA from the cytoplasm of mouse cells, was studied. Sequence analysis of its tryptic peptides revealed that the RNA-linked protein is identical to prothymosin alpha. Very similar RNA-protein complexes were identified in human, bovine and yeast cells. Tryptic peptide maps of 125I-labelled RNA-linked proteins of diverse origin demonstrated their marked similarity, thus indicating high evolutionary conservation of prothymosin alpha from yeast to man.

Amino Acid Sequence↗
PubMed2001

Oversynthesis of a new Escherichia coli small RNA suppresses export toxicity of DsbA'-PhoA unfoldable periplasmic proteins.

In Escherichia coli, the DsbA'-PhoA hybrid proteins carrying an unfoldable DsbA' fragment can be targeted to the envelope, where they exert their toxicity. Hybrid proteins stick to the periplasmic face of the inner membrane and paralyze the export mechanism, becoming lethal if sufficiently overproduced and if not degraded by the DegP protease (A. Guigueno, P. Belin, and P. L. Boquet, J. Bacteriol. 179:3260-3269, 1997). We isolated a multicopy suppressor that restores viability to a degP strain without modifying the expression level of the toxic fusion. Suppression does not involve activation of the known envelope stress-combative pathways, the Cpx pathway and the sigma(E) regulon. Subclone analysis of the suppressor revealed a 195-bp DNA fragment that is responsible for toxicity suppression. The cloned gene, called uptR, is approximately 130 bp long (including the promoter and a transcription termination signal) and is transcribed into a small RNA (92 nucleotides). Using site-directed mutagenesis, we found that UptR RNA does not require translation for toxicity suppression. UptR-mediated action reduces the amount of membrane-bound toxic hybrid protein. UptR RNA is the first example of a small RNA implicated in extracytoplasmic toxicity suppression. It appears to offer a new way of suppressing toxicity, and its possible modes of action are discussed.

Alkaline Phosphatase↗
PubMed2006

Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA.

MPSS (massively parallel signature sequencing) is a sequencing-based technology that uses a unique method to quantify gene expression level, generating millions of short sequence tags per library. We have created a series of databases for four species (Arabidopsis, rice, grape and Magnaporthe grisea, the rice blast fungus). Our MPSS databases measure the expression level of most genes under defined conditions and provide information about potentially novel transcripts (antisense transcripts, alternative splice isoforms and regulatory intergenic transcripts). A modified version of MPSS has been used to perform deep profiling of small RNAs from Arabidopsis, and we have recently adapted our database to display these data. Interpretation of the small RNA MPSS data is facilitated by the inclusion of extensive repeat data in our genome viewer. All the data and the tools introduced in this article are available at http://mpss.udel.edu.

Arabidopsis↗