Nucleic Acids Research, 2011, Vol. 39, No. 1 285 Figure 3. Impact of bystin or hTsr1 depletion on ribosome biogenesis. (A) Cytoplasmic fractions, prepared from cycloheximide-treated cells transfected for 48 h either with a scramble siRNA or a siRNA targeting bystin or hTsr1, were separated on sucrose gradients by ultracentrifugation. (B) Identical amounts of RNAs (3 mg) from HeLa cells, extracted 48 h after transfection, were separated on a 1% agarose gel and then transferred to a nylon membrane. The pre-rRNA species were revealed by hybridization with various 32P-labeled probes (50ITS1, an equimolar mixture of ITS2b and ITS2d/e, or a mixture of 18S and 28S probes). Pre-rRNAs are described in Figure 1, except the 26S species which extends from site A0 to 2. (C) The labeling of each band was quantiﬁed with a phosphorimager and normalized to the amount of 28S RNA measured in the corresponding lane. The means of six different samples in three independent northern blots are presented in the histogram as variation rates relative to control cells. Pair-wise statistical analysis was performed with Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Northern blot analysis with the 50ITS1 probe shows presence of the 21S-C species in nuclear RNAs from control HeLa cells. complementary to the 50-ETS/ETS1, the ITS1 or the ITS2 (Figure 5). Cells treated with bystin-speciﬁc siRNAs over-accumulated 41S and 21S pre-rRNAs, while the amount of 18S-E pre-RNA decreased (Figure 3B and 3C). The build-up of 41S pre-rRNA was also evidenced with the ITS2 probes, while the 32S and 12S large subunit precursors were unaffected compared with control cells. These pre-rRNA patterns indicate delayed cleavage in ITS1 at site 2 and deﬁcient maturation of the 21S pre-rRNA within the pre-40S particles. In addition, we observed the conspicuous build-up of a shorter form of the 21S pre-rRNA, that we named 21S-C (court is the French for short). Such a form was previously described upon depletion of RPS19, and named 20S pre-rRNA (43); however this nomenclature is confusing considering the 20S pre-rRNA in S. cerevisiae. Careful examination of total RNAs’ northern blot proﬁles showed the reproducible presence of a shoulder in the 21S peak at this position in control cells, suggesting that the 21S-C is a regular processing intermediate that accu- mulates upon bystin depletion (data not shown). Indeed, this intermediate could be clearly detected in the nuclear fraction of control cells (Figure 3D). Additional probes were used to determine the 30-end of this species by northern blot (Figure 4A). Both the 21S and 21S-C pre-rRNAs were revealed with probes spanning position 170 (ITS1-160) and 615 (ITS1-605) in the ITS1. In contrast, probes hybridizing around nucleotides 650 (ITS1-639) or 730 (ITS1-721) no longer revealed the 21S-C RNA. Considering that at least 15 nt are required for speciﬁc hybridization of these oligonucleotidic probes in our experimental conditions, these results indicate that the 30 extremity of the 21S-C pre-rRNA is roughly located between nucleotides 620 and 655 of the ITS1 (Figure 4B). If the 21S-C pre-rRNA resulted from an alternative endonucleolytic cleavage in the ITS1, one would expect the appearance of a complementary cleavage product.