Altered subcellular localization of mutant RPS19 characterized in 2 DBA patients. A DBA patient carried the mutation Gly127Gln located in the C-terminal NoS, and another one carried a double missense mutation, Val15Phe + Thr55Met, in which the Val15Phe was located in the first NoS defined in this study. In each panel (A-C, E) the left field shows DAPI nuclear staining, the middle field shows FITC staining of the GFP-RPS19 fusion proteins, and the right field shows merged fields of DAPI + FITC staining. We analyzed the subcellular distribution of RPS19 after transfection of Cos-7 cells with either a GFP-mutated Gly127Gln (panel A), Val15Phe + Thr55Met (panel B), or Val15Phe (panel C) RPS19. Each DBA mutant: Gly127Gln and Val15Phe + Thr55Met impaired RPS19 nucleolar import (panels A-B). In the double mutant, the missense mutation Val15Phe is solely responsible for the impairment of RPS19 nucleolar import since Val15Phe RPS19 failed to localize to the nucleoli (panel C), while Thr55Met RPS19 exhibited normal nucleolar localization (data not shown). Arrows indicate the absence of RPS19 nucleolar staining in the Cos-7 cell shown. (D) In order to rule out a putative effect of the fixation with chilled methanol, RPS19 subcellular distribution was investigated in live transfected cells. The left panel depicts the bright field illumination, and the right panel shows the fluorescent GFP-RPS19 fusion protein. Arrows mark RPS19 nucleolar localization. (E) Green fluorescent fusion protein GFP-WT RPS19 (left panel, FITC staining) and the red fluorescent fusion protein DsRed1-mutated Val15Phe RPS19 (middle panel, Texas red staining) were coexpressed in transfected Cos-7 cells. Normal RPS19 was localized to the nucleoli, while mutant Val15Phe (red) was not imported into the nucleoli (right panel, DAPI + FITC + Texas red staining). Original magnifications: × 63 (A-C,E); × 40 (D).