<i>In vitro</i> processing of 20S pre-rRNA is impaired in the <i>rpl3</i>[W255C] mutant.

<p><i>In vitro</i> cleavage assays were performed with pre-ribosomal particles purified <i>via</i> PTH-tagged Nob1 from different strains: wild-type (blue, circle), <i>rpl3</i>[W255C] (red, square), <i>rpl3</i>[K30E] (green, square) and <i>rsa3Δ</i> (purple, triangle). Purified particles were incubated in reaction buffer containing 1 mM ATP (A and B) or 1 mM GTP (C and D) for the indicated times (0, 2, 5, 10 and 30 min). RNA was extracted and cleavage at site D was analysed by primer extension with probe c' (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004205#pgen.1004205.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004205#pgen.1004205.s011" target="_blank">Table S3</a>). Representative primer extension analyses are shown (A and C). The strong upper stops result from termination at sites of 18S rRNA base-dimethylation at A₁₇₈₁ and A₁₇₈₂. These modifications precede site D cleavage <i>in vivo</i>. The black arrow indicates site D. Filled and empty dots indicate non-relevant primer extensions stops that were observed in some experiments (for further discussion, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004205#pgen.1004205-Lebaron1" target="_blank">[26]</a>). Signal intensities were measured by phosphoimager scanning; values were corrected for RNA loading using the dimethylation signals as internal standards, normalised to the sample at the zero time-point, arbitrarily set at 1.0, and plotted (B and D). The average of 2 (B) and 4 (D) independent experiments is shown; the error bars indicate the standard deviation.