West Nile virus genome harbors essential riboregulatory elements with conserved and host-specific functional roles
Ontology highlight
ABSTRACT:
ORGANISM(S): Chlorocebus aethiops Aedes albopictus
PROVIDER: GSE228446 | GEO | 2024/07/16
REPOSITORIES: GEO
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Project description:Six transcripts of the gene SERPINA1 (serpin family A member 1) were sequenced via SHAPE-MaP to obtain their secondary structure profiles. For each transcript, SHAPE-MaP includes a sample treated with 1M7 reagent ('p'), a minus reagent negative control ('m'), and a denatured control ('d'). The 1M7 reagent preferentially reacts with unpaired bases in RNA and subsequently induces mutations during the reverse transcription step of library preparation. After sequencing and alignment, the 'mutational profiles' of the 'p', 'd' and 'm' samples are used to calculate the SHAPE reactivity of each base in the transcript. SHAPE reactivity correlates to the tendency of a base to be paired as part of a secondary structure.
2016-11-01 | GSE81525 | GEO
Project description:A SHAPE-MaP structure probing experiment was performed on 39 firefly luciferase mRNAs containing uracil, 1-methyl-psuedouracil, or 5-methoxy-uracil. For each mRNA, SHAPE-MaP includes a sample treated with 1M6 ('MOD'), a minus reagent ('NC'), and a denatured control ('DEN'). The 1M6 reagent preferenctially reacts with unpaired bases in RNA and subsequently induces mutations during the reverse transcription step of library preparation. After sequencing and alignment, the 'mutational profiles' of the 'p', 'm', and 'd' samples are used to calculate the SHAPE reactivity of each base.
2019-10-22 | GSE139176 | GEO
Project description:Due to the mounting evidence that RNA structure plays a critical role in regulating almost any essential physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 performs comparably well to NAI on naked RNA under in vitro conditions and it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.
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Project description:Spliceosomal snRNA are key components of small nuclear ribonucleoprotein particles (snRNPs), the building blocks of the spliceosome. The biogenesis of snRNPs is a complex process involving multiple cellular and subcellular compartments, the details of which are yet to be described. In short, the snRNA is exported to the cytoplasm as 3‘-end extended precursor (pre-snRNA), where it acquires a heptameric Sm ring. The SMN complex which catalyses this step, recruits Sm proteins and assembles them around the pre-snRNA at the single stranded Sm site. After additional modification, the complex is re-imported into the nucleus where the final maturation step occurs. Our modeling suggests that during the cytoplasmic stage of maturation pre-snRNA assumes a compact secondary structure containing Near Sm site Stem (NSS) which is not compattible with the formation of the Sm ring. To validate our in silico predictions we employed selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) on U2 snRNA in vivo, ex vivo and in vitro, and U4 pre-snRNA in vitro. For the in vivo experiment HeLa cells were incubated for 10 min at 37°C with NAI or DMSO to final concentration 200 mM. RNA was isolated using Trizol (Sigma) and 200 µl chloroform and precipitated with ethanol at -20°C overnight. For the ex vivo experiment, RNA was isolated from HeLa cells after Protease K treatment at room temperature for 45 min. After incubation, RNA was isolated using equilibrated phenol/chloroform/isoamyl alcohol buffered by folding buffer (110 mM HEPES pH 8.0, 110 mM KCl, 11 mM MgCl2) and cleaned on a PD-10 column according to the manufacturer’s instructions. Isolated RNA was treated with 100mM NAI or DMSO for 10 min at 37°C. For the in vitro experiment, U2WT and U4 pre-snRNA were transcribed by T7 polymerase followed by DNase I (30 min at 37 °C) and Proteinase K (30 min at 37°C) treatments. U2 snRNA was purified on 30 kDa Amicon columns, folded for 30 min at 37°C in 57 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. U4 pre-snRNA was purified on Superdex 200 Increase 10/300GL, folded for 30 min at 37°C in 60 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. All prepared RNA samples (in vitro, ex vivo, in vivo) were used for reverse transcription with the gene-specific primer 5’-CGTTCCTGGAGGTACTGCAA for U2 snRNA and 5’- AAAAATTCAGTCTCCG for U4 pre-snRNA. We used SHAPE MaP buffer (50 mM Tris-HCl pH 8.0, 75 mM KCl, 10 mM DTT, 0.5 mM dNTP, 6 mM MnCl2) and SuperScript II (Invitrogen). Amplicons for snRNAs were generated using gene-specific forward and reverse primers. Importantly, the primers include Nextera adaptors required for downstream library construction. PCR reaction products were cleaned using Monarch PCR&DNA Clean-up Kits. Remaining Illumina adaptor sequences were added using the PCR MasterMix and index primers provided in the NexteraXT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were quantified using Qubit (Invitrogen) and BioAnalyzer (Agilent). Amplicons were sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit. All sequencing data was analyzed using the ShapeMapper 2 analysis pipeline1. The ‘—amplicon’ and ‘—primers’ flags were used, along with sequences of gene-specific handles PCR primers, to ensure primer binding sites are excluded from reactivity calculations. Default read-depth thresholds of 5000x were used. Analysis of statistically significant reactivity differences between ex vivo and in vivo-determined SHAPE reactivities was performed using the DeltaSHAPE automated analysis tool and default settings2. 1. Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143-148 (2018). 2. Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10, 1643-69 (2015).
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Project description:DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. In human genomes, an epigenetic modification at the fifth carbon of cytosine bases comprises one fundamental pathway by which genes can be silenced or activated . Methods widely used to detect epigenetic modifications at cytosine bases rely on either deamination of unmodified cytosines to read as thymine or borane reduction of the modified oxidised cytosine bases to read as thymine. As a result, such methods fail to capture common C-to-T mutations, and importantly also fail to distinguish 5-methylcytosine (5mC - mainly found in silenced parts of the genome) from 5-hydroxymethylcytosine (5hmC - enriched in active gene bodies and enhancers). Hence, existing methods are unable to read the complete information stored in our genomes in a single workflow. Here, we build on biomodal's duet multiomics solution +modC, which has the unique ability to read genetic and epigenetic cytosine modifications (modC) in a single workflow. By introducing a high-fidelity methyltransferase during the workflow, we are able to construct an expanded information table that can be used to deconvolute A,T,C,G, 5-mC and 5-hmC simultaneously in a single read within a DNA molecule . We use a mouse embryonic stem cell-line, ES-E14TG2A, to map for the first time a simultaneous reading of the genome and epigenome at high depth and show how these epigenetic modifications are segregated across the genome. In summary, using our modified approach we demonstrate simultaneous, phased reading of all six genetic and epigenetic bases. This tool provides a more complete picture of the information stored in genomes and will have applications throughout biology and medicine.
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