Sequence variants within the 3′-UTR of the COL5A1 gene alters mRNA stability: Implications for musculoskeletal soft tissue injuries

Abstract

COL5A1 encodes the α1 chain of type V collagen, a quantitatively minor fibrillar collagen that regulates fibrillogenesis. A variant within the 3′-UTR of COL5A1 is associated with chronic Achilles tendinopathy (AT) and other exercise-related phenotypes but the functional significance of this is unknown. The aim of this study was therefore to identify functional differences between the COL5A1 3′-UTR from patients with AT and asymptomatic controls. To this end we have used a reporter assay in which the COL5A1 3′-UTR from AT patients and controls were cloned downstream of the firefly luciferase gene and luciferase activity measured as an indication of mRNA stability. When the cloned COL5A13′-UTRs were sequenced, two major forms named C- and T-alleles were predominantly identified in the controls and the AT subjects respectively. The luciferase activity of the C-alleles was significantly lower than that of the T-alleles (69.0 ± 22.0% (N = 24) vs. 90.6 ± 13.7% (N = 30), p < 0.001) which suggests an overall increase in mRNA stability for the T-allele. Furthermore, we identified a functional miRNA site for Hsa-miR-608 within the COL5A1 3′-UTR and using deletion constructs we have identified additional elements which regulate COL5A1 mRNA stability. These results have important implications for our understanding of the molecular basis of musculoskeletal soft tissue injuries and other exercise-related phenotypes.

Introduction

Collagen fibrils are the basic building blocks of fibrous connective tissues such as tendons, ligaments and cartilage, as well as the extracellular matrix (ECM) of other tissues such as skeletal muscle (Kadler et al., 2007). Type I and III collagens are the most abundant protein in non-cartilaginous fibrils. Although quantitatively minor, the type V collagen isoforms, which are heterotrimers made up of various combinations of the α1(V), α2(V) and α3(V) chains, are also critical structural components of non-cartilaginous fibrils (Chanut-Delalande et al., 2004, Kadler et al., 2007). Within non-cartilaginous tissues, type V collagen intercalates with the type I and III collagens to form heterotypic fibrils, where it plays an important role in fibril assembly and lateral growth (Birk, 2001). The triple helical portion of type V collagen is embedded in the fibril while the amino-terminal globular domain projects onto the surface (Birk et al., 1990). The major isoform of type V collagen contains two α1(V) and one α2(V) chains, which are encoded by the COL5A1 and COL5A2 genes, respectively (Wenstrup et al., 2004, Malfait et al., 2010).

Disease-causing mutations have been identified in about half of classical (types I and II) Ehlers–Danlos syndrome (EDS) patients and mutations within the COL5A1 and COL5A2 genes have been identified in about 46% and 4% of these patients, respectively (Malfait et al., 2010, Wenstrup and De Paepe, 2010). Furthermore, expression from both copies of COL5A1 is required for normal fibrillogenesis. Loss of function mutations within one copy of COL5A1 (haploinsufficiency) is common in EDS patients (Wenstrup et al., 2006, Malfait et al., 2010) and is related to the presence of large irregular collagen fibrils in their connective tissue (Vogel et al., 1979). Whereas similar findings have been reported in Col5a1^+/− mice, Col5a1^−/− mice die in utero emphasising that type V collagen is an essential protein (Wenstrup et al., 2006). The α2(V) chain is also involved in regulating fibrillogenesis. Mice harbouring a functional mutation in the amino-cleavage domain of the Col5a2 gene predominately produce an α1(V) homotrimer, which is not incorporated in the dermal heterotypic type I collagen fibrils. This in turn negatively affects the organisation of the matrix, reduces the thickness of the basement membranes in the epidermis and increases apoptosis of the stromal fibroblasts (Chanut-Delalande et al., 2004).

Recently, a common C/T single nucleotide polymorphism (SNP rs12722; also known as the BstUI RFLP) in the 3′-untranslated region (3′-UTR) of COL5A1 was shown to be associated with chronic Achilles tendinopathy (Mokone et al., 2006, September et al., 2009) and with anterior cruciate ligament (ACL) injuries in females (Posthumus et al., 2009). In all these studies the CC genotype was shown to be over-represented in the respective asymptomatic controls. An age-related increase in range of motion measurements has also been reported in individuals with the CC genotype (Brown et al., 2011).

Interestingly, generalised joint hypermobility is one of the clinical features of EDS (Malfait et al., 2010), while both a decrease and increase in flexibility have been reported to be a modifiable risk factor for Achilles tendon injuries (Kaufman et al., 1999) and ACL ruptures (Uhorchak et al., 2003). Despite a growing body of evidence that indicates a genetic association of sequence variants within the COL5A1 3′-UTR and complex phenotypes (Mokone et al., 2006, Collins et al., 2009, Posthumus et al., 2009, September et al., 2009, Posthumus et al., 2011, Brown et al., 2011, Brown et al., in press), the biological function, if any, of the COL5A1 3′-UTR is currently unknown.

Using several SNPs which spanned the 3′-UTR, we have previously mapped the region within the COL5A1 3′-UTR which was associated with chronic Achilles tendinopathy (September et al., 2009). The associated region not only contained the BstUI RFLP, but two additional SNPs in close proximity to two putative microRNA (miRNA) binding sites (September et al., 2009). These results are interesting because elements within the 3′-UTR of eukaryotic genes are emerging as important post-transcriptional regulators and have been shown to be involved in the aetiology of many diseases (Mazumder et al., 2003, Garzon et al., 2009). In particular, miRNAs, short non-coding RNAs between 18 and 24 nucleotides, are capable of repressing protein synthesis by modulating the stability and/or the translational efficiency of target mRNAs by binding to their 3′-UTR (Garzon et al., 2009).

The aim of this study was to test whether the COL5A1 3′-UTR cloned from patients with chronic Achilles tendinopathy and asymptomatic controls were functionally different. Furthermore, the possible biological function of the putative miRNA binding sites within the immediate area associated with Achilles tendinopathy was also examined (Fig. 4).