Ontology highlight
ABSTRACT:
PROVIDER: PRJNA762079 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15881370_1.fastq.gz | Fastqsanger.gz | |||
| SRR15881370_2.fastq.gz | Fastqsanger.gz | |||
| SRR15881371_1.fastq.gz | Fastqsanger.gz | |||
| SRR15881371_2.fastq.gz | Fastqsanger.gz | |||
| SRR15881372_1.fastq.gz | Fastqsanger.gz |
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Project description:Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
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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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Project description:Single-molecule correlated chemical probing of RNA
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Project description:We identified a SNP rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple ChIP-seq experiments, and its strong-binding allele increased transactivation, and accorded higher mRNA levels in cell lines and human brain. To confirm the allele-specific binding, we conducted ChIP tagmentaion sequencing experiment in human lymphoblastoid cell line GM12763 which is heterozygous for rs242561. NRF2 ChIP DNA was isolated from GM12763 cells treated with Sulforaphane (SFN) in triplicates. The region containing rs242561 was amplified using primers, Fwd 5â??-AGCCTTCCCTGTCCTTGATT-3â??, Rev 5â??-GGACCGAGCTTCCAGTCTAA-3â??, and tagmentated using Tn5-based transposition for library construction. Libraries were sequenced on Illumina MiSeq. NRF2 ChIP tagmentation sequencing of a 241bp intron region in MAPT gene using GM12763 treated with sulforaphane
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Project description:The nuclear envelope serves as important mRNA surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualised nuclear export in trypanosomes by probing large, endogenous mRNA by intramolecular multi-colour single molecule FISH (smFISH). In addition, unspliced mRNAs were visualised by co-probing two adjacent introns or intergenic regions. We found that the initation of nuclear export requires neither the completion of transcription nor trans-splicing. Nevertheless, the inhibition of trans-splicing blocked cytoplasmic transport of the of unspliced mRNAs and only a small fraction reached the nucleus-distant cytoplasm. Most of the unspliced transcripts remained at the nuclear periphery, within transport and in nuclear periphery granules (NPGs) localised at the cytoplasmic site of nuclear pores that resemble stress granules in composition. Our work shows that, in striking contrast to other eukaryotes, trypanosomes can start nuclear export while the mRNA is still transcribed, but unspliced transcripts remain ‘stuck’ in nuclear pores, probably awaiting processing or decay. Our data indicate that trypanosomes regulate the completion of nuclear export rather than the start.
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