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High throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

ABSTRACT: High throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

PROVIDER: PRJNA483770 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7629020_1.fastq.gz	Fastqsanger.gz
	SRR7629020_2.fastq.gz	Fastqsanger.gz
	SRR7629021_1.fastq.gz	Fastqsanger.gz
	SRR7629021_2.fastq.gz	Fastqsanger.gz
	SRR7629022_1.fastq.gz	Fastqsanger.gz

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High throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

Project description:These data were obtained using a recently-developed high throughput method to probe regional RNA accessibility by mimicking in vivo antisense hybridization. The method (INTERFACE) is an engineered RNA system (for use in E. coli) that exploits conserved bacterial mechanisms of translational stalling and Rho-dependent transcription termination mechanisms to quantify RNA hybridization (via an asRNA targeting an RNA region of interest) via a transcriptional elongation response. We first establish the high throughput system by evaluating the accessibility profile of a well-characterized gI intron (overexpressed). We then apply this method with bioinformatics and machine learning approaches to profile over 900 RNA-interaction interfaces in 71 validated, but largely mechanistically under-characterized, sRNAs of Escherichia coli, in the presence and absence of a global regulator, Hfq. Importantly, we showcase the utility of INTERFACE in detecting RNA regions whose hybridization is Hfq-sensitive, finding two thirds of tested sRNAs with Hfq dependency. Further, we identify in vivo hybridization patterns that hallmark functional regions in 16 well-characterized sRNAs and couple these insights to a computational target prediction algorithm to identify novel mRNA targets for six under-characterized and two well-characterized sRNAs.

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High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites.

Project description:Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.

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