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.

mTORC1 phosphorylates LARP6 to stimulate type I collagen expression

“…7). Based on our previous finding that LARP6 interacts with Sec61810, we postulate that in the absence of S348/S409 phosphorylation LARP6 has a diminished ability to dissociate from the ER membrane. This prevents its shuttling into the nucleus and participation in a new round of collagen mRNA metabolism111314.…”

“…In one dimensional SDS-PAGE (1DGE), endogenous LARP6 migrated as two bands (Fig. 1a, arrows), representing differently phosphorylated LARP6 isoforms10. The slower migrating band was markedly reduced by mTOR inhibitors (Fig.…”

“…We have previously reported that LARP6 tethers collagen mRNAs to the ER membrane, where it interacts with Sec61810. Therefore, we assessed if the interaction of wt HA-LARP6 and S348A/S409A with Sec61 correlates with their accumulation in the microsomal fraction.…”

“…Compelling evidence has shown that collagen expression is primarily regulated at the posttranscriptional level, including regulation of half-life and translation of collagen mRNAs4567. Binding of RNA binding protein La ribonucleoprotein domain family, member 6 (LARP6) to the conserved structural element in the 5′UTR of collagen α1(I) and α2(I) mRNAs (5′ stem-loop) (5′SL) regulates their translation891011. LARP6 tethers collagen mRNAs to the cytoskeletal filaments; nonmuscle myosin and vimentin912.…”

La-related protein 6 controls ciliated cell differentiation

“…pExpress1 X. laevis mcidas (accession number BC124892) was used for the synthesis of mcidas mRNA. Other plasmids for WISH probes including sox2 and mcidas or for microinjection including human LARP6 (HA-hL6), Δ1-300, Δ1-478, Δ135Δ264 and LARP6-GFP (L6-GFP) were described in previous publication [12, 15, 21, 22]. …”