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Fig. 5 A very short form of 5.8S rRNA contributes to ribosome heterogeneity in RPL17+/mut cells. (A) Sequence of the human 5.8S rRNA with the 2 canonical 5′ ends and that of mouse 5.8SC rRNA previously determined (54), with the positions of the probes used in this figure. (B) 5.8S rRNA species separated on a 6% polyacrylamide gel (bottom) were identified with the 5.8S probe hybridizing to the core of the 5.8S rRNA, or with the 5′-5.8S probe complementary to the 5′-end of the 5.8S (A). The 7SK RNA was used as a loading control. (C) The ratios of the 5.8S rRNA species to 7SK snRNA (B) were quantified. Values were normalized to the mean value obtained for controls and are displayed as log2 values. (D) LCLs from unaffected individual 1-II-5 were treated with 3 siRNAs targeting RPL17 mRNA. Total RNA profiles analyzed on agarose (top) or polyacrylamide gels (bottom) were compared to those of untreated cells (–) or cells treated with a scramble siRNA. (E) Cytoplasmic fractions extracted from control (black) or case (gray) lymphoblastoid cell lines were analyzed by ultracentrifugation on 10%–50% sucrose gradients. Total RNAs from the gradient fractions were extracted and analyzed by Northern blot with the 5.8S probe.