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HuR targetome analysis in THP-1 macrophages using HuR FLASH-Seq.

ABSTRACT: HuR targetome analysis in THP-1 macrophages using HuR FLASH-Seq.

PROVIDER: PRJNA1070260 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27754127_1.fastq.gz	Fastqsanger.gz
	SRR27754127_2.fastq.gz	Fastqsanger.gz
	SRR27754128_1.fastq.gz	Fastqsanger.gz
	SRR27754128_2.fastq.gz	Fastqsanger.gz
	SRR27754129_1.fastq.gz	Fastqsanger.gz

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HuR targetome analysis in THP-1 macrophages using HuR FLASH-Seq.

Project description:FLASH, a novel method, was utilized to identify binding sites of RNA-binding proteins (RBPs) with single-nucleotide resolution (Nucleic Acids Res 2020;48:e15). In order to identify HuR target genes in THP-1 macrophages after circARCN1 regulation, we performed FLASH experiments using the anti-HuR antibody on THP-1 macrophages with circARCN1 knockdown or overexpression. The resulting FLASH products were then subjected to RNA sequencing.

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Project description:Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the post-transcriptional level. HuR, ELAVL1, is an RNA binding protein that binds and regulate the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS induced response. Here we investigated to what extent small molecules inhibiting HuR-RNA interaction, called tanshinone mimics, counteract the LPS induced macrophage response. We show that tanshinone mimics are present in solution in a keto-enolic tautomerism and that, by molecular dynamic calculations, the ortho quinone form is the preferred species interacting with HuR and favoring the closure of its conformation to a no-binding mode. The protection of the enolic status with diacetate caused the loss of activity of tanshinone mimics in vitro but active in vivo. Murine macrophages cell line RAW264.7 was treated with LPS and tanshinone mimics and the modulation of the LPS induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes and that tanshinone mimics reduced the interaction. Functional annotation addressed a specific set of genes, as Cxcl10, Il1b, Cd40, Fas, involved in chemotaxis and immune response whose association with HuR decreased and led to a reduction of their protein level and secretion. The same effect was observed in primary murine bone marrow derived macrophages and in vivo in a LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing tanshinone mimics with anti-inflammatory properties in vivo.

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