Structure-guided mutagenesis of SmD3. (A) Stereo view of the human U1 snRNP structure highlighting the fold of SmD3 (depicted as a cartoon trace with magenta β strands and cyan helices) and its interactions with neighboring subunits U1-C/Yhc1 (green) and U1-70K/Snp1 (blue) and with the Sm site in U1 snRNA. The zinc atom nucleating the U1-C/Yhc1 fold is depicted as a magenta sphere. Selected amino acids are shown as stick models and numbered according to their positions in the yeast polypeptides. Atomic contacts are indicated by dashed lines. (B) Alignment of the primary structures of the S. cerevisiae (Sce) and human (Hsa) SmD3. Positions of side-chain identity/similarity are indicated by • above the alignment. The secondary structure elements are depicted above the alignment, with β strands as magenta arrows and α helices as cyan cylinders. SmD3 amino acids that make contacts to other U1 snRNP subunits or contact the U1 snRNA are highlighted in color-coded boxes as indicated. Reverse arrowheads indicate the boundaries of the C terminal truncations of yeast SmD3; black and red arrowheads denote viable and lethal truncations, respectively. (C) The wild-type and truncated SMD3 alleles were tested for activity by plasmid shuffle in smd3▵, smd3▵ mud2▵, smd3▵ nam8▵, smd3▵ mud1▵, and smd3▵ tgs1▵ strains. The viable FOA-resistant smd3▵ strains bearing the indicated SMD3 alleles were spot-tested for growth on YPD agar at the temperatures specified. SMD3 alleles listed at the bottom of each panel failed to complement smd3▵ in the plasmid shuffle assay and were deemed lethal in that genetic background.
