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Targeted DMS probing of SF3A3 5' UTR

ABSTRACT: Targeted DMS probing of SF3A3 5' UTR

PROVIDER: PRJNA615008 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11411712.fastq.gz	Fastqsanger.gz
	SRR11411713.fastq.gz	Fastqsanger.gz
	SRR11411714.fastq.gz	Fastqsanger.gz
	SRR11411715.fastq.gz	Fastqsanger.gz
	SRR11411716.fastq.gz	Fastqsanger.gz

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Project description:To delineate the native structure of SF3A3 5'UTR, RNA was harvested from IMR90 human fibroblasts. Using specific primers and DMS-MaPSeq pipeline, we validated individual base pairing probabilities within the endogenous 5'UTR of SF3A3 (samples described as 'in vivo' transcribed). DMS-MaP-Seq is based on the principle that DMS is highly reactive to solvent-accessible, unpaired adenine (A) and cytosine (C) residues, but remains inert toward base-paired A and C engaged in Watson-Crick interactions (Rouskin et al., 2014). Using this methodology, we identify stable stem-loop structure (SL3) positioned within SF3A3 5'UTR. To further validate the functional importance of SL3, the structural point mutant (SF3A3 5'UTR mut: A55C and U95A) and rescue (SF3A3 5'UTR res: A55C and U95A and rescuing point mutations G61U and U100G) sequences of SF3A3 5'UTR were cloned into the reporter plasmid. For the validation of these mutate-and-rescue constructs, plasmids were in vitro transcribed and either used directly (samples described as 'in vitro') for DMS-MaP-Seq probing.

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