Collagens are the most abundant proteins in the human body, they are homo or heterotrimeric protein complexes that form higher order structures such as fibrils and networks in the extracellular matrix. They as such, play crucial roles in maintenance of animal tissues integrity and remodeling during growth, differentiation as well as wound healing. Vertebrates have at least 27 collagen types and several thousand mutations in collagen subunit coding genes have been reported that are linked to human diseases (for a review see Myllyharju J. and Kivirikko KI., (2004) Trends in Genet, 20:33-43 and references therein). Type I collagen structural disorders cause Osteogenesis Imperfecta (an inherited bone fragility disorder) ( http://www.le.ac.uk/ge/collagen/ ), while up-regulated deposition of type I collagen is the hallmark of fibroproliferative disorders of highly deleterious impact to the human health.
Over the past years, work conducted by B.Stefanovic and collaborators (Florida State University, Tallahasee, USA) identified the human LARP6 protein as a regulatory switch to type I collagen production. Through a serie of four articles, B.Stefanovic and coll. not only provide a first model of the translational regulation of type I collagen production but also emphasises the importance of HsLARP6 function in human health (Cai L. et al (2010a,b); Challa A. and Stefanovic B. (2011), Manojlovic Z. and Stefanovic B. (2011)).
LARP6 is a member of LARP family of proteins, function of which has been poorly characterized. Our recent work has shown that human LARP6 (HsLARP6) regulates translation of mRNAs encoding type I collagen. Type I collagen is the most abundant protein in human body and its expression is highly upregulated in fibroproliferative disorders. The protein is composed of two α1(I) chains and one α2(I) chain, which are encoded by different mRNAs. Collagen α1(I) mRNA and α2(I) mRNA have a unique 5’ stem-loop structure (5’SL) in their 5’ untranslated regions. HsLARP6 binds collagen 5’ SL with high affinity and specificity. 5’SL has a central bulge surrounded by two stems. The single stranded regions of the bulge of 5’SL have 9 nucleotides, but only 5 nucleotides are involved in binding HsLARP6. The affinity of binding of HsLARP6 to 5’SL was estimated to be 2-5 nM. No any other high affinity binding target of HsLARP6 has been identified. Knock down of HsLARP6 or mutation of 5’SL in collagen α1(I) mRNA greatly reduced collagen expression. Knock-in mice, which have a mutation in collagen α1(I) gene that disrupted the 5’SL, are resistant to development of liver fibrosis. These findings suggest that binding of HsLARP6 to 5’SL is critical for high expression of type I collagen and development of the fibrosis in vivo.
Several proteins interact with HsLARP6 and contribute to regulation of collagen expression. These proteins include RNA helicase A (RHA), nonmuscle myosin and vimentin. RHA is required to unwind structural elements in mRNAs to facilitate translation initiation. RHA does not directly bind collagen mRNAs, but is recruited to the 5’SL by interaction with HsLARP6. When RHA is knocked down, polysomes can not efficiently assemble on collagen mRNAs and they are poorly translated. This suggests that RHA is recruited by HsLARP6 to unwind the 5’SL and facilitate translation of collagen mRNAs.
HsLARP6 also interacts through its La domain with intermediate filaments composed of vimentin. This interaction associates collagen mRNAs with vimentin filaments. In situ RNA hybridization and immunostaining for vimentin revealed co-localization of collagen mRNAs with the filaments. Cells in which vimentin filaments have been disrupted or cells from vimentin knock-out mice have reduced level of collagen α1(I) and α2(I) mRNAs, because the half-life of collagen mRNAs in these cells is reduced by more than 50%. This suggests that association of collagen mRNAs with vimentin stabilizes these mRNAs. Thus, HsLARP6, not only regulates translation of collagen mRNAs by recruiting RHA, but also regulates their stability by binding them to the intermediate filaments composed of vimentin.
HsLARP6 also interacts with another type of filaments, those composed of nonmuscle myosin. Nonmuscle myosin has always been implicated in cell motility and contractility. However, disruption of nonmuscle myosin filaments results in greatly diminished collagen synthesis. Nonmuscle myosin is found associated with polysomes, indicating a possible role in translation. Immunoprecipitation of nonmuscle myosin pulls down collagen mRNAs and HsLARP6. At present, it is not clear how nonmuscle myosin regulates collagen expression. Our hypothesis is that HsLARP6 targets collagen mRNAs to the filaments composed of nonmuscle myosin and that integrity of these filaments is necessary for translation initiation on collagen mRNAs. Thus, by switching the binding from vimentin filaments to nonmuscle filaments, HsLARP6 regulates if the collagen mRNAs will be stored and stabilized or if they will be committed to translation.
All these roles clearly established HsLARP6 as the key regulator of synthesis of the most abundant protein in human body, type I collagen.
Branko Stefanovic (College of Medicine, Florida State University , Tallahassee, USA)
(Manojlovic Z. and Stefanovic B., 2011)
(Challa, A. and Stefanovic B., 2011)
