Down-regulation of RPS19 expression blocks maturation of the 18S rRNA. (A) HeLa cells were transfected with either rps19 or scramble siRNAs and cultured for 24 hours or 48 hours. After RNA extraction, the mRNAs encoding the ribosomal proteins RPS19 and P0 were amplified by RT-PCR. The β-actin mRNA was coamplified with RPS19 as an internal control. RT-PCR reactions obtained from untreated cells (control) and Oligofectamine-treated cells are also shown. The 3 siRNAs (rps19-1, rps19-2, rps19-3) differ in their efficacy to down-regulate the RPS19 mRNA level. (B) Knockdown of RPS19 expression upon treatment with siRNA rps19-1 is confirmed by Western blot analysis of total cell extracts. Densitometry shows that up to 90% of the protein is depleted 72 hours after transfection. (C) Analysis on sucrose gradient of the ribosomal subunits from HeLa cells 48 hours after transfection with the rps19-1 or scramble siRNAs shows that depletion of RPS19 strongly affects 40S subunit production, leading to a strong imbalance between the free 60S subunits and 40S subunits. (D) Northern blot analysis of total RNA extracts from cells transfected with rps19 siRNAs using oligonucleotides hybridizing in the mature 18S and 28S rRNAs. The 18S and 28S rRNA levels were evaluated by phosphoimager quantification. (E) Northern blot analysis with probes complementary to the ITS1 (5′-ITS1) or to the ITS2. “Control” indicates untreated cells; “Oligofect.,” transfection treatment without siRNAs; “scrbl,” scramble siRNA. All experiments were repeated at least 3 times with similar results.