Figure 4.
DDX21 is required for access of the late-acting SNORD56 and SNORD68 to pre-40S intermediates. (A-C) Extracts from cells transfected with siRNAs targeting DDX21 (siDDX21), with or without expression of RNAi-resistant wild-type DDX21 or inactive DDX21 (DDX21SAT), as well as extracts from cells treated with non-target siRNAs (siNT) or those targeting PWP2 (siPWP2), were separated by sucrose gradient density centrifugation. RNA was isolated from gradient fractions and analysed by denaturing polyacrylamide gel electrophoresis followed by Northern blotting for the snoRNAs SNORD100 (A), SNORD68 (B) and SNORD56 (C). Quantifications of three independent experiments are shown above the Northern blots for each fraction as percentage of the total snoRNA signal (data are presented as mean ± SEM); fractions containing free snoRNPs and ribosomal complexes are indicated below. (D) The ratio of SNORD68 and SNORD56 signals in pre-40S-containing fractions to those containing free snoRNPs in (B) and (C) was calculated, normalized to the ratio in the corresponding sample transfected with non-target siRNAs and plotted as mean ± SEM.