Background/Purpose: Persistent myofibroblast activation and associated excessive extracellular matrix protein deposition are hallmarks of systemic sclerosis (SSc). However, the mechanisms that account for this excessive fibrotic response remain elusive, hampering development of targeted treatment modalities. Alternative polyadenylation (APA) allows adding poly(A) tail to different polyadenylation sites which gives rise to transcripts with variable 3’ UTR length. Cleavage factor Im 25 (CFIm25 – gene name: Nudt21) has been recently identified as a key regulator of APA ¹. CFIm25 deletion leads to 3’ UTR shortening of key genes involved cell fate determination. A shortened 3’ UTR will often lack microRNA binding sites in comparison to its long form, resulting in increased mRNA translation due to evasion of microRNA-mediated gene repression. The goal of this study is to understand the role of CFIM25 and APA in SSc pathogenesis.

Methods: CFIm25 expression and cellular localization was investigated in skin from SSc patients and murine models. RNA sequencing was carried out using control or CFIm25 knockdown fibroblasts to determine CFIm25 targets, which were subsequently verified by Real-time PCR and Western Blot. Finally, bleomycin was administrated subcutaneously in mice with conditional CFIm25 deletion in fibroblasts to determine the effect of CFIm25 repression in vivo.

Results: CFlm25 mRNA was downregulated in SSc skin compared to matched controls (Fig 1A). Patients with disease duration < 2 years had significantly lower CFIm25 mRNA levels than the remainder of patients (Fig 1B). Moreover, downregulation of CFIm25 in SSc skin was primarily observed in myofibroblasts in IHC experiments.

Global RNA sequencing experiments upon knockdown of CFIm25 in normal skin fibroblasts (n=5 per group) resulted in 3’ UTR shortening of 971 genes. TFG-β was predicted to be the top upstream regulator of this gene list.  Moreover, CFIm25 depletion led to enhanced protein expression of key fibrotic genes, including COL1A1 and TGFBR1. Similarly, these genes showed 3’UTR shortening in SSc skin compared to matched controls (n=10 per group).

CFIm25 protein levels were decreased in the skin of bleomycin dermal fibrosis and Tight Skin Mouse I murine models. Finally, conditional CFIm25 deletion in fibroblasts led to exaggerated skin fibrosis upon bleomycin treatment (n=10 per group), as measured by Masson’s trichrome staining, collagen I and TGFBR1 Western Blot (Fig 1C), and dermal thickness (Fig 1D).

Conclusion: We link for the first time a recently discovered key RNA processing protein, CFIm25 to 3’ UTR shortening and increased transcription/translation of TGF-β regulated profibrotic genes, in dermal fibrosis models and SSc. CFIm25 rescue can be a potential therapeutic target in SSc.

1.       Brumbaugh J. et al. Cell. 2017