Rpl35A is required for pre-rRNA processing in ITS1 and ITs2. (A) A schematic of human pre-rRNA processing. Mature ribosomal RNA species are indicated by shaded boxes: ■, 18S; , 5.8S; □, 28S. External and internal transcribed spacers are indicated as lines between the mature species and labeled above the primary pre-rRNA transcript. Cleavage sites, as originally proposed by Hadjiolova et al,⁵³  are shown by numbered arrows above the 45S and 45S′ transcripts. The sequence of cleavage of the 45S′ pre-RNA at sites 1 and 2 results in 2 alternative processing pathways. Two additional human cleavage sites (2b and 4a) shown as numbered arrows below the transcript are inferred from these studies. The presence of a 7S precursor to 5.8S rRNA implies an additional cleavage (4a) within ITS2. An additional cleavage site corresponding to the yeast A3 site (2b) within ITS1 is also proposed. The positions of oligonucleotide probes used for Northern analysis are shown in gray below the primary transcript. (B,C) Northern analysis of rRNA from RPL35A knock-down in UT7-Epo demonstrates steady-state increases in 45S:32S and 32S:12S ratios, indicating a disruption of 32S pre-rRNA maturation with resultant decreases in mature 28S (B) and 5.8S (C) rRNA. The rRNA species are indicated to the right of each panel; the probe used is indicated in gray to the left of each panel. Eth indicates ethidium bromide; Sh-Luc, Luciferase-control transduced cells; sh-1, -2, -3, or -4, respective RPL35A shRNAs.