Collagen mRNAs are inefficiently translated in the absence of RHA. (A) Polysomal profile of HEK293 cells with and without RHA. (Top panel) Cells were transfected with RHA-specific siRNA or control siRNA and polysomes fractionated on linear sucrose gradients (fraction 1, 45% sucrose; fraction 16, 15% sucrose; fractions 16 and 17, postpolysomal supernatant). (Top panel) The OD260 of the fractions from cells transfected with siRNAs (RHA) (dotted line) and control siRNA(Sc) (full line) is shown. (Bottom panel) Distribution of ribosomal RNA in the fractions. Fractions containing polysomes are indicated. (B) Knockdown of RHA by siRNA. Control siRNA (Sc, lane 1) and RHA-specific siRNA (RHA, lane 2) were transfected into HEK293 cells, and expression of RHA was analyzed by Western blot. Loading control: tubulin (TUB). (C) RHA distribution in polysomal fractions of HEK293 cells. (Top panel) Sucrose fractions as in A were probed for the presence of RHA by Western blot. (Bottom panel) The fractions were analyzed by Western blot for LARP6. (D) Polysomal loading of collagen α1(I) mRNA is RHA-dependent. Polysomes were fractionated from HEK293 cells transfected with control siRNAs (Sc, top panel) or RHA-specific siRNA (RHA, bottom panel), and the fractions were analyzed for collagen α1(I) mRNA by radiolabeled RT-PCR. (E) Polysomal loading of collagen α2(I) mRNA is RHA-dependent. Same experiment as in C, except collagen α2(I) was analyzed. (F) Knockdown of RHA does not affect polysomal loading of GAPDH mRNA. Same experiment as in C, except GAPDH mRNA was analyzed.
