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ABSTRACT: In vivo and Ex vivo SHAPE-MaP structure probing of human lncRNA MEG3

PROVIDER: PRJNA552583 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9641660_1.fastq.gz	Fastqsanger.gz
	SRR9641660_2.fastq.gz	Fastqsanger.gz
	SRR9641661_1.fastq.gz	Fastqsanger.gz
	SRR9641661_2.fastq.gz	Fastqsanger.gz
	SRR9641654_1.fastq.gz	Fastqsanger.gz

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Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [SHAPE-Map]

Project description:To refine the authentic CENP-C binding sites of lnc-CCTT and globally map lnc-CCTT secondary structure, we also performed SHAPE-MaP (selective 2’-hydroxyl acylation analyzes by primer extension and mutational profiling), which uses hydroxyl-selective electrophiles to modify the 2’-hydroxyl groups of unbound single-stranded nucleotides, in HeLa cells both ex vivo and in vivo. Lnc-CCTT secondary structure was modeled by combination SHAPE data from cell-free ex vivo with pairing probabilities. As expected, nucleotides 43-79 nt, a determinant for RNA-DNA triplex formation, exhibited a continuous single-strandedness, which may be prone to binding DNA. More importantly, only nucleotides 118-177 nt, which was folded into a stem-loop structure in the secondary structure, showed a significant reduced SHAPE reactivities in cell when comparing to cell-free state, suggesting this region could be attributed to interaction with protein components.

2022-04-20 | GSE149529 | GEO

SHAPE analysis of RNA structure in living cells with unprecedented accuracy

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.

2020-12-16 | GSE154563 | GEO

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Project description:Ex vivo and In-Cell Mouse Xist SHAPE-MaP

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The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation

Project description:The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain’s imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5’-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

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mRNA structure regulates protein expression through changes in functional half-life

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.

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Complete in vivo SHAPE-MaP structure of the SARS-CoV-2 genome

Project description:SHAPE-MaP structure probing experiment was performed on SARS-CoV-2 infected Vero cells at 4 days post infection with two biological replicates. For each replciate, SHAPE-MaP includes a sample treated with 2-methylnicotinic acid imidazolide acid (modified) or a minue reagent (unmodified). NAI preferentially reacts with unpaired bases in RNA, forming acylated bases. These modifications are encoded as mutation during reverse transcripatse and library preparation. After sequencing and alignment, the reactivity profiles of 'modified' and 'unmodified' samples are used to calculate SHAPE reactivity of each base

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Project description:SHAPE-MaP structure probing experiment was performed on SARS-CoV-2 infected Vero or C6/36 cells at 4 days post infection with two biological replicates. For each replciate, SHAPE-MaP includes a sample treated with 2-methylnicotinic acid imidazolide acid (modified) or a minuse reagent (unmodified). NAI preferentially reacts with unpaired bases in RNA, forming acylated bases. These modifications are encoded as mutation during reverse transcripatse and library preparation. After sequencing and alignment, the reactivity profiles of 'modified' and 'unmodified' samples are used to calculate SHAPE reactivity of each base

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ChIP-seq in MRC5 hIPSCs overexpressing MEG3 RNA.

Project description:This experiment was designed to detect differences in PRC2 occupancy in a human induced pluripotent cell line (MRC5) naturally deficient in MEG3 RNA after lentiviral overexpression of MEG3. MRC5 were transduced with lentiviruses for the overexpression of MEG3 lncRNA or GFP as a control. ChIP-seq was performed with EZH2 and JARID2 antibodies.

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SHAPE and DMS probing of SLNCR1 lncRNA conserved region 403-780

Project description:The goal of this study was to understand the underlying structure of nucleotides 403-780 of the lncRNA SLNCR1, in-cell and when extracted from nuclear and cytoplasmic fractions. SHAPE and DMS probing revealed that the region is largely unstructured inside and outside of the cell, and appears protein-bound in primary melanoma cells.

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PAIR-MaP visualizes RNA base pairing complexity in human and bacterial non-coding RNAs

Project description:Structure probing experiments were performed on in vitro transcripts and E. coli and human cell cultures under natively extracted (cell-free) and in-cell conditions to benchmark the performance of the newly introduced PAIR-MaP correlated chemical probing strategy for detecting RNA duplexes. Multiple-hit dimethyl sulfate (DMS) probing was done using new buffer conditions that facilitate DMS modification of all four nucleotides.

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