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Stiff matrix instigates type I collagen biogenesis by mammalian cleavage factor I complex-mediated alternative polyadenylation
Zijing Zhou, … , Ping Chen, Yong Zhou
Zijing Zhou, … , Ping Chen, Yong Zhou
Published January 14, 2020
Citation Information: JCI Insight. 2020;5(3):e133972. https://doi.org/10.1172/jci.insight.133972.
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Research Article Pulmonology

Stiff matrix instigates type I collagen biogenesis by mammalian cleavage factor I complex-mediated alternative polyadenylation

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Abstract

Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3′ UTR cleavage and polyadenylation represents a common characteristic among many disease states, including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I–mediated (CFIm-mediated) APA in regulating extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulated expression of CFIm68, CFIm59 and CFIm25 subunits and promoted APA in favor of the proximal poly(A) site usage in the 3′ UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix–induced APA and overproduction of COL1A1, whereas CFIm did not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits was associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3′ UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.

Authors

Zijing Zhou, Jing Qu, Li He, Yi Zhu, Shan-Zhong Yang, Feng Zhang, Ting Guo, Hong Peng, Ping Chen, Yong Zhou

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Figure 5

Effects of knockdown of CFIm68 on matrix stiffness–regulated APA and expression of COL1A1 and FN1.

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Effects of knockdown of CFIm68 on matrix stiffness–regulated APA and exp...
(A) CFIm68-specific siRNA (si-68) and the control siRNA (si-con) were transfected into human lung fibroblasts. Knockdown of CFIm68 expression was determined by immunoblot. GAPDH was used as loading control. (B–G) Lung fibroblasts transfected with si-68 and si-con were cultured on soft and stiff hydrogels for 48 hours. Ratios of long-to-total COL1A1 (B) and FN1 (E) transcripts were used to evaluate the proximal poly(A) site usage. Relative levels of COL1A1 (C) and FN1 (F) mRNA were determined by real-time RT-PCR. Protein levels of COL1A1 (D) and FN1 (G) were determined by immunoblot. GAPDH was used as internal reference or loading control. Bar graphs represent (mean ± SD) 3 or 4 separate experiments. Statistical analysis was performed by 1-way ANOVA.
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