Bioinformatics analysis suggests base modifications of tRNAs and miRNAs in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Modifications of RNA bases have been found in some mRNAs and non-coding RNAs including rRNAs, tRNAs, and snRNAs, where modified bases are important for RNA function. Little is known about RNA base modifications in <it>Arabidopsis thaliana</it>.</p> <p>Results</p> <p>In the current work, we carried out a bioinformatics analysis of RNA base modifications in tRNAs and miRNAs using large numbers of cDNA sequences of small RNAs (sRNAs) generated with the 454 technology and the massively parallel signature sequencing (MPSS) method. We looked for sRNAs that map to the genome sequence with one-base mismatch (OMM), which indicate candidate modified nucleotides. We obtained 1,187 sites with possible RNA base modifications supported by both 454 and MPSS sequences. Seven hundred and three of these sites were within tRNA loci. Nucleotide substitutions were frequently located in the T arm (substitutions from A to U or G), upstream of the D arm (from G to C, U, or A), and downstream of the D arm (from G to U). The positions of major substitution sites corresponded with the following known RNA base modifications in tRNAs: N1-methyladenosine (m¹A), N2-methylguanosine (m²G), and N2-N2-methylguanosine (m²₂G).</p> <p>Conclusion</p> <p>These results indicate that our bioinformatics method successfully detected modified nucleotides in tRNAs. Using this method, we also found 147 substitution sites in miRNA loci. As with tRNAs, substitutions from A to U or G and from G to C, U, or A were common, suggesting that base modifications might be similar in tRNAs and miRNAs. We suggest that miRNAs contain modified bases and such modifications might be important for miRNA maturation and/or function.</p

The performance of blended conventional and novel binders in the in-situ stabilisation/solidification of a contaminated site soil.

This paper presents an investigation of the effects of novel binders and pH values on the effectiveness of the in-situ stabilisation/solidification technique in treating heavy metals and organic contaminated soils after 1.5-year treatment. To evaluate the performance of different binders, made ground soils of SMiRT site, upto 5 m depth, were stabilised/solidified with the triple auger system and cores were taken for laboratory testing after treatment. Twenty four different binders were used including Portland cement (PC), ground granulated blastfurnace slag (GGBS), pulverised fuel ash (PFA), MgO and zeolite. Unconfined compressive strength (UCS), leachate pH and the leachability of heavy metals and total organics were applied to study the behaviours of binders in treating site soils. Under various contaminant level and binder level, the results show that UCS values were 22-3476 kPa, the leachability of the total organics was in the range of 22-241 mg/l and the heavy metals was in the range of 0.002-0.225 mg/l. In addition, the combination of GGBS and MgO at a ratio of 9:1 shows better immobilisation efficiency in treating heavy metals and organic contaminated soils after 1.5-year treatment, and the binding mechanisms under different binders were also discussed in this paper.This work was supported by the UK Technology Strategy Board (Project TP/5/CON/6/I/H0304E) and the Schlumberger Foundation.F. Wang et al. Journal of Hazardous Materials 285 (2015) 46–52, http://dx.doi.org/10.1016/j.jhazmat.2014.11.002 NOTICE: this is the author’s version of a work that was accepted for publication in the Journal of Hazardous Materials. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in the Journal of Hazardous Materials, [285, 46-52, (2015)] 10.1016/j.jhazmat.2014.11.00

ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis.

Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2

Multiple distinct small RNAs originate from the same microRNA precursors

Abstract Background MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes. Results We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes. Conclusions We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought

Blockade of Renin Angiotensin System Increased Resistance to STZ-Induced Diabetes in Rats with Long-Term High-Fat Diet

This study aimed to investigate whether rennin-angiotensin system (RAS) blockade through telmisartan would increase the resistance to streptozotocin- (STZ-) induced diabetes in insulin resistance rats. There were sixty Wistar rats that were divided into four groups: normal control (NC), high-fat diet (HF), high-fat diet plus STZ injection (HF+S), and high-fat diet plus STZ injection and telmisartan intervention (HF+S+T). Five rats were chosen randomly and respectively from groups NC and HF to undergo a hyperinsulinemic euglycemic clamp. Another five rats were selected randomly from the four groups, respectively, for intravenous injection insulin releasing test (IVIRT), and the other five rats for pancreas specimens used in islet cell immunohistochemistry staining (stained for insulin, NF-κB, and caspase-3), islet cell apoptosis staining, and reverse transcription PCR (AT1R and IL-1 beta). There was a significant difference of overt diabetes incidence between groups HF+S+T and HF+S (P<0.05). Furthermore, inflammatory markers and islet cell apoptosis were found to be significantly reduced in group HF+S+T compared with group HF+S (all P<0.01 or P<0.05). Overall, telmisartan-treated rats were found to have reduced RAS activity, increased resistance to STZ-induced diabetes, reduced inflammatory markers, and improvement of islet cell function and morphology

siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plant