Type I collagen is the most abundant protein in human body, produced by folding of two α1(I) and one α2(I) polypeptides into the triple helix. A conserved stem-loop structure is found in the 5' UTR of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family, member 6 (LARP6) as the protein which binds the collagen 5' stem-loop in the sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain, not found in other members of the La superfamily. LARP6 interacts with the two single stranded regions of 5' stemloop. The Kd for binding of LARP6 to the 5' stem-loop is 1.4 nM. LARP6 binds the 5' stem-loop in both, the nucleus and cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes, however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by siRNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum (ER). We could reproduce this focal pattern of synthesis using collagen/GFP reporter protein, but only when the reporter was encoded by the mRNA with the 5' stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in absence of LARP6, it accumulated diffusely throughout the ER. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that LARP6 dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.

Type I collagen is the most abundant protein in human body synthesized in all tissues as the heterotrimer of two α1(I) and one α2(I) polypeptides. Here we show that intact nonmuscle myosin filaments are required for synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds RNA binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion collagen α1(I) homotrimer, in diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides, which is required for folding of the heterotrimer, and in their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes and there is a subset of polysomes involved in myosin dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating translation of collagen mRNAs.

Hepatic stellate cells (HSCs) are mesenchymal cells of the liver, activation of which is responsible for excessive synthesis of extracellular matrix, including type I collagen, and development of liver fibrosis. The activation of HSCs is driven by transcription factors and pair-related homeobox transcription factor Prx1 was identified as one of the transcription factors involved in this process, because transcription of collagen alpha1(I) gene is stimulated by Prx1 in HSCs and in the liver. Here, we show that expression of the RNA-binding protein RBMS3 is upregulated in the activation of HSCs and fibrotic livers. Immunoprecipitation followed by differential display identified Prx1 mRNA as one of the mRNAs interacting with RBMS3. The RBMS3 sequence-specific binding site was mapped to 60 nt located 1946 nt 3' of the stop codon of Prx1 mRNA. Ectopic expression of RBMS3 in quiescent HSCs, which express trace amounts of type I collagen, increased expression of Prx1 mRNA and collagen alpha1(I) mRNA. Expression of reporter Prx1 mRNA containing the RBMS3 binding site was higher than the mRNA lacking this site. Over-expression of RBMS3 further increased the steady-state level of the reporter mRNA-containing RBMS3 binding site, but had no effect on the mRNA lacking this site. Binding of RBMS3 to the Prx1 3' UTR increased the half-life of this mRNA, resulting in increased protein synthesis. These results suggest that RBMS3, by binding Prx1 mRNA in a sequence-specific manner, controls Prx1 expression and indirectly collagen synthesis. This is the first description of the function of RBMS3, as a key regulator of profibrotic potential of HSCs, representing a novel mechanism by which activated HSCs contribute to liver fibrosis.

Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3–dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells.

No abstract