The Role of the 3′UTR Region in the Regulation of the ACVR1/Alk-2 Gene Expression

Abstract

BackgroundThe ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3′UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression.ResultsThe ACVR1/Alk-2 3′UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3′UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3′UTR sequence thereby modulating its expression.ConclusionIn this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3′UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3′UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.

Signaling Receptors for TGF-β Family Members

“…In differentiating adipocytes, miR-30c is up-regulated and silences ALK-2 expression (Karbiener et al 2011); miR-148b and miR-365 target the 3 0 -untranslated region of ALK-2 mRNA (Mura et al 2012); down-regulation of ALK-2 mRNA by miR-148a is relevant for the cancer stem-cell population in hepatocellular carcinoma (Li et al 2015); in the liver, BMP signaling is coordinated with the homeostasis of iron; and, after depletion of iron levels, miR-130a expression is up-regulated causing ALK-2 silencing and decrease in BMP-6 signaling, leading to down-regulation of the BMP target gene hepcidin, which produces the liver hormone that regulates iron homeostasis (Zumbrennen-Bullough et al 2014).…”

MicroRNAs and post-transcriptional regulation of skeletal development

“…miR-210 positively regulates osteoblast commitment by targeting the Acvr1b receptor (type 1 receptor; Mizuno et al 2009). The 3 0 -UTR of the ACVR1/ALK2 gene has recently been studied in vitro to elucidate miRNAs that when expressed induce BMP signaling alterations in fibrodysplasia ossificans progressiva (Mura et al 2012). Several additional BMP-modulated miRNAs have been identified (Li et al 2008, 2009a, Lin et al 2009, Bae et al 2012, Gamez et al 2013, generally as a result of high-throughput expression analysis but without precise information on their transcriptional control and function.…”

Towards a cure for Fibrodysplasia ossificans progressiva

“…This has been followed up using in vitro and in vivo models of HO, altogether demonstrating a link between an overactivation of the FOP receptor and enhanced BMP signaling, eventually leading to HO. This concept has focused the research in FOP in the subsequent years, aiming to identify novel approaches to prevent overactive ALK2 signaling by means of small molecule inhibitors targeting the kinase activity of the receptor (7-9) or anti-sense oligonucleotides, including small interference RNAs (siRNA) (10), antisense oligonucleotides (AONs) (11) and micro RNAs (miRNAs) (12) to inhibit mutant ACVR1 expression (see Figure 1). …”