Effects of Rpl17 and Rpl10 knockdowns on translation and ribosome biogenesis. (A) Depletion of Rpl17 and Rpl10 results in distinct alterations in polysome profiles. Cells stably transfected with doxycycline-inducible shRNAs against Rpl17 and Rpl10 were treated with doxycycline for 48 h or remained untreated (+Dox, −Dox). Cell lysates were centrifuged through 10%–45% (w/w) sucrose gradients and fractionated with the continuous measurement of absorbance at 254 nm. Halfmers are marked with red arrowheads. (B) RAMP profiles of Rpl17 and Rpl10 knockdowns showing changes in steady-state levels of pre-rRNA intermediates 48 h after siRNA transfections. Total cellular RNA was extracted and analyzed by northern hybridizations, ratios of various rRNA intermediates in knockdowns were normalized to the corresponding ratios in control cells transfected with nontargeting siRNA (i.e., log2 = 0 indicates no change relative to control cells; see Wang et al. [2014] for further details on this assay). (PTP) Primary transcript plus, early 47–45S pre-rRNAs. (C) RT-PCR analysis of Rpl17, Rpl10, and Xrn2 knockdowns; Rpl23a was used as a reference gene. (D) Primer extension to examine levels of 28.5S pre-rRNA, a short-lived product of ITS2 cleavage at site 4b. (E) Schematic representation of the mouse 47S pre-rRNA transcript with cleavage sites and major pre-rRNA processing intermediates indicated. (ETS) External transcribed spacer; (ITS) internal transcribed spacer. (F) Knockdown of Rpl17 or Rpl10 severely inhibits cell proliferation. Cells were transfected with nontargeting siRNA (Ctrl), or siRNAs against Rpl17 and Rpl10 siRNA. Cell numbers were determined in triplicate transfections; error bars indicate SD. (G) Depletion of Rpl17 and Rpl10 results in altered 36S and 12S pre-rRNA levels and accumulation of pre-rRNA decay products (asterisk). Total RNA was analyzed by a northern hybridization at 48 h after transfection with the indicated siRNAs.
