Identification of HuR-specific RNA interactome in TGF-ß1-activated fibroblasts in response to cigarette smoke

Abstract

Aims and objectives: Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the associated mechanism remains unknown. Our aim was to study the RNA-regulatory networks of the stress-responsive RNA-binding protein HuR and what impact it has on the disease pathology.

Methods: TGF-β1-activated human lung fibroblasts were used to study the effect of cigarette smoke extract (CSE) stimulation on the translocation HuR from the nucleus to the cytoplasm. HuR-specific RNA-interactions in the nuclear and cytoplasmic cell fractions were analyzed by CLIP (cross-linking and immunoprecipitation) and subsequent high-throughput sequencing.

Results: We showed that CSE induces HuR translocation from the nucleus to the cytoplasm in TGF-β1-activated lung fibroblasts. CLIP data revealed several fibrotic genes, including COL1A1 and COL1A2, among the most frequently bound HuR-targets in the cytoplasm. Evaluation of HuR-bound RNA sequences showed a difference in 4-meric binding motifs between nuclear and cytoplasmic HuR fractions, with increased binding to polyU-rich RNA motifs in the cytoplasm. Interestingly, the polyU-rich binding was reduced upon TGF-β1/CSE stimulation. Additionally, the TGF-β1/CSE stimulation led to reduced binding of cytoplasmic HuR to 3’UTR RNA regions and increased interaction with the coding regions.

Conclusions: Our data suggests a global shift of HuR function from 3’UTR-dependent RNA stabilization, potentially in the direction of translational regulation of genes associated with fibrosis.