Figure 7.

Primer extension analysis and Northern-blot probing of pre-rRNA maturation intermediates for U3 variants mutated in helices I, II, III and their linking segments. (A) Target sites on the pre-rRNA of oligonucleotides used for northern-blotting. The positions of the probe target sequences are indicated with black squares. Probe 001 was used to detect the entire 35S pre-rRNA, the 33S RNA only cleaved at site A0 and the 27S A2 and 27S A3 intermediates containing the pre-rRNA sequences upstream of sites A2 or A3. Probe 013 was used for RNAs 27A2/A3 and 27B which 3′ extremities corresponds to sites A2, A3 and B, respectively. Probe 007 allowed evaluation of the amount of mature 25S rRNA. Probe 003 was used to detect 23S RNA cleaved at site A3 without cleavage at A0 and the aberrant accumulation of 22S RNA cleaved at site A0 and A3 without cleavage at sites A1 and A2. Probe 002 also detected the accumulation of 22S RNA, the aberrant apparition of an A0-A2 intermediates, and the 20S RNA, the normal last intermediate before 18S production. Probe 026 detected the aberrant 22S and A0-A2 intermediates as well as an aberrant 19S RNA in extracts of cells expressing the variant 3-2 and 3-3 U3 RNAs. Finally, probe 008 was used to detect mature 18S rRNA in Northern-blot experiments and to detect 5′-termini A0 and A1 in primer extension analyses, as described in Figure 6G. (B) Primer extension analyses with primer 008 performed on 2 μg of total RNA extracted from JH84 cells expressing WT or mutant U3 snoRNAs: (–) control assays with an empty plasmid; for other lanes, the identity of the U3 snoRNA expressed in the cells is indicated above the autoradiograms. Longer exposure time was used for detection of pre-rRNA intermediates cleaved at site A0 (22S and A0–A2 intermediates) than for detection of products cleaved at A1 (20S intermediates and mature 18S rRNA). (C, D) Autoradiograms of Northern-blot analyses performed on total RNA extracted from JH84 cells expressing WT or mutant U3 snoRNAs after growth on YPD medium. RNA were fractionated by electrophoresis in 1.2% agarose-6% formaldehyde gels. The parts of interest in the autoradiogram obtained for each probes are shown as distinct sub-panels, identified by the number of the probe used, indicated on the side (see Supplementary Table S1 for probe sequences). The identities of the U3 snoRNAs variants used in the experiments are given on top of the series of autoradiograms. Panel C corresponds to U3 mutated in segment 3 (variants 3-1, 3-2, 3-3 and 3–5). The first two lanes correspond to WT U3 and no U3 expression (–), respectively. Panel D corresponds to U3 mutated in linking segment 1 (1-1) and mutants of segments III, II and I, the latter previously proposed to form helix I. The pre-rRNA maturation intermediates identified by Northern-blot and their schematic representation are indicated on the right (C) or between (D) the panels.