Rpl35A is required for large ribosomal subunit assembly and pre-rRNA processing. (A) To identify potential sources of altered proliferation, polysome analysis was performed on UT-7/Epo (left column) and HEK293A (right column) cells infected with control (top row) or RPL35A (bottom row) sh-1. Cells were sorted 3 days after lentiviral infection and returned to media for 24 hours before treatment with cycloheximide and fractionation of lysates on a sucrose gradient. The 40S, 60S, and 80S peaks are indicated by; the polysome fraction lies below the horizontal line. In comparison to control shRNA-infected cells, cells infected with RPL35A shRNA demonstrated a decreased 40S:60/80S ratio, indicating a relative reduction of free 60S subunits. Similar results were seen in UT-7/Epo and HEK293A cells transduced with RPL35A sh-3 (not shown). Experiments were performed once with 2 different RPL35A shRNA for HEK293A cells and twice with 2 RPL35A shRNAs in UT-7/Epo cells. (B) Metabolic labeling of nascent RNA with ³²P was used to identify abnormalities of pre-rRNA processing. Lentivirus-infected cells sorted 6 days after infection were plated in phosphate-free media in 6-well plates for 2 hours before the addition of ³²P orthophosphate for 1 hour, washed, and then incubated in complete media for 4 hours. RNA was fractionated on 1.3% agarose/formaldehyde gels, dried, and autoradiographed. denotes the indicated mature and pre-rRNA species. RPL35A knockdown resulted in marked decrease of 32S, 28S, and 12S labeling without affecting mature 18S labeling. An increased exposure of the gel demonstrates reduced 7S and 5.8S rRNA. An ethidium bromide stain of the gel is also shown. (C) Intensity profile of lanes 1 and 2 demonstrates the reduction of 28S, preservation of 18S, and the appearance of a 41S band (shoulder adjacent to the 45S peak) not seen in control cells. Eth indicates ethidium bromide; sh-Luc, Luciferase-control transduced cells; sh-1, -2, -3, and -4, respective RPL35A shRNAs.