Defects in Rrp12 function block the synthesis of 40S subunits but not of 60S subunits.

<p>(<b>A</b>) Structure of the 35S pre-rRNA and major intermediates of the rRNA processing pathway. The names of the initial pre-rRNA species are highlighted in brown. Those for the 18S pre-rRNA precursor, 5.8S/25S precursors, and 5′-A₀ processing byproduct are highlighted in blue, green and grey, respectively. For simplicity, an alternative pathway to form 27SB_L pre-rRNA is not shown. Binding sites for oligonucleotide probes (01 to 08) used in Northern blot experiments are indicated in the upper diagram. Those included probe 03 for the DA₂ region, probe 04 for the A₂–A₃ region, probe 01 for the 5′-A₀ region, probe 02 for the 18S region, probe 07 for the E-C₂ region, probe 08 for the 25S region, probe 06 for the 5′EC₂ region and probe 05 for the 5.8S region. (<b>B</b>) Sucrose-gradient sedimentation analysis of ribosomal fractions (40S, 60S, 80S and polysomes) of cell lysates from <i>GAL::HA-rrp12</i> cells that have been grown in galactose (Gal)-containing media or shifted to a glucose (Glu)-containing media for 9 hours. Depletion of Rrp12 protein was analyzed by Western blot (<b>C</b>). (<b>D</b> and <b>E</b>) Northern blot (NB) analysis of total RNAs extracted from <i>RRP12</i>, <i>GAL::HA-rrp12</i>, and <i>GAL::HA-rrp12</i> cells containing plasmids encoding either wild type Rrp12 or the hypomorphic Rrp12 (deletion Δ1-198) mutant. Cells were grown at 30°C in galactose-containing media and shifted to glucose-containing media for the indicated times. The specific region of the 35S pre-rRNA recognized by the Northern blot probe is indicated on the right. This will be similarly indicated in the rest of analyses presented in this work. The thin white lines between lanes 6 and 7 indicate the presence of in-between lanes in the same blot that have been removed. The experiment shown in E also includes, as a loading control, the RNA of the signal recognition particle scR1. (<b>F</b>) Sucrose-gradient sedimentation analysis of ribosomal fractions (40S, 60S, 80S and polysomes) of cell lysates from <i>GAL::HA-rrp12</i> cells containing plasmids encoding either wild type Rrp12 or the hypomorphic Rrp12 (Δ1-198) mutant. Cells were grown continuously in glucose-containing media.