title: Characterization of binding of LARP6 to the 5' stem-loop of collagen mRNAs: implications for synthesis of type I collagen.
abstract: Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5' UTRs, the 5'SL. LARP6 binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5'SL of collagen α2(I) mRNA is more stable than the binding to 5'SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5'SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5'SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5'SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis.
authors.affiliation: a Department of Biomedical Sciences; College of Medicine ; Florida State University ; Tallahassee , FL USA.
authors.forename: Lela
authors.initials: L
authors.lastname: Stefanovic
authors.forename: Liam
authors.initials: L
authors.lastname: Longo
authors.forename: Yujie
authors.initials: Y
authors.lastname: Zhang
authors.forename: Branko
authors.initials: B
authors.lastname: Stefanovic
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D003094:Collagen / Q000235:genetics* / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D000069816:SEC Translocation Channels; D017386:Sequence Homology, Amino Acid; D000097585:SS-B Antigen
chemical_list: D020121:5' Untranslated Regions; D001324:Autoantigens; D003094:Collagen; D024042:Collagen Type I; D008565:Membrane Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D020038:RNA, Spliced Leader; D012261:Ribonucleoproteins; D000069816:SEC Translocation Channels; D000097585:SS-B Antigen
keywords: LARP6; RNA stem-loop; fluorescence polarization; mRNA binding; type I collagen