RHA is required for efficient synthesis of collagen protein. (A) Knockdown of RHA decreases collagen expression. HEK293 cells were transfected with control siRNA (Sc, lane 1) or with two RHA-specific siRNAs (RHA, lane 2), and the expression of RHA, collagen α1(I) polypeptide, and tubulin was analyzed by Western blot. (B) Rescue of collagen expression by the siRNA-resistant RHA gene (RHA*). RHA was knocked down by RHA-specific siRNAs (RHA, lanes 1,2) or the cells were transfected with control siRNA (Sc, lane 3). The siRNA-resistant RHA construct (RHA*) was then transfected (lane 2), or the cells were untransfected (lanes 1,3). Collagen α1(I), RHA, and tubulin expression was measured by Western blot. (C) Knockdown of RHA does not affect expression of collagen mRNAs. Total RNA from the experiment in A was analyzed for expression of collagen α1(I), collagen α2(I), and GAPDH mRNA by quantitative real-time RT-PCR. Relative expression of collagen α1(I) and collagen α2(I) mRNAs was standardized by the internal control GAPDH. Error bars, ±1 SEM.
