Fig. 2
A conserved loop in Rps20 promotes cytoplasmic 40S subunit maturation. a–c Crucial cytoplasmic pre-40S maturation steps are functionally interconnected. A RIO2 (rio2Δ) shuffle ltv1Δ strain (a), a RIO2 (rio2Δ) RPS3 (rps3Δ) double shuffle strain (b), and a RIO2 (rio2Δ) RPS20 (rps20Δ) double shuffle strain (c) were transformed with plasmids carrying the indicated wild-type and mutant alleles. Transformants were spotted in ten-fold serial dilutions on SDC-Leu-Trp (-leu-trp) or 5-FOA containing medium (to select for cells that have lost the respective URA3 plasmid(s) harboring the wild-type gene(s)) and growth was monitored after incubation at 30 °C for the indicated days. d Rps20 loop reaches through the pre-40S subunit toward Rio2 on the intersubunit side. Rps20 (green), Rps3 N-domain (red), and Rio2 (blue) in the pre-40S structure (PDB 6FAI)20 (upper panel). Sequence alignment reveals conservation of the Rps20 loop between eukaryotes and archaea (lower panel). Arrows point to residues R68, K69, E74, and K77, which were mutated in this study. e, f Rps20 loop is crucial for cell growth. An RPS20 (rps20Δ) shuffle strain was transformed with the indicated plasmid-based alleles and transformants were spotted in ten-fold serial dilutions on SDC-Leu and SDC+5-FOA plates (e) or, after plasmid shuffling on 5-FOA, on YPD plates (f). Growth was monitored after incubation at 30 °C for the indicated days. g–i Rps20 loop mutants strongly impair 40S subunit synthesis. g Polysome profiles of the indicated rps20 loop mutants were recorded after centrifugation on 7–45% sucrose gradients. h Northern blot analyses of total RNA extracts from wild-type RPS20 or the indicated rps20 mutant cells using probes detecting the 20S pre-rRNA, mature 18S rRNA, and 25S rRNA (left panel). Simplified pre-rRNA processing scheme (right panel). All detected (pre-)rRNA species are indicated in bold letters. The binding site of the D/A2 probe used to detect the 20S pre-rRNA and precursors thereof is indicated in magenta. i Cells expressing wild-type RPS20 or the indicated rps20 alleles were analyzed by fluorescence in situ hybridization (FISH) using a Cy3-labeled probe specific to the D/A2 segment of internal transcribed spacer 1 (ITS1; depicted in h). The nucleoplasm was stained with DAPI. PC phase contrast. Scale bar is 5 µm
