FIGURE 6.
Association of Tif6p and Hrr25p with pre-66 S ribosomal particles. The RNA isolated from the TAP-Tif6p-purified complex as well as from whole cell-free extracts (as described under “Experimental Procedures”) was subjected to Northern analysis (A) using 32P-labeled deoxyoligonucleotide probes complementary to different regions of 35 S pre-rRNA, shown in the lower panel of A and B, that specifically detects each pre-rRNA intermediate. B, primer extension analysis was carried out to detect 35 S and 25.5 S pre-rRNAs. +, cells expressing TAP-tagged Tif6p; –, cells expressing untagged wild-type cells. A schematic diagram of the processing intermediates of 35 S pre-rRNA to mature 25 S and 18 S rRNAs is also shown. After autoradiography by phosphorimaging, the band intensity of each RNA species was calculated using the ImageQuant software. Relative enrichment of each species was calculated as described by Harnpicharnchai et al. (10). This consisted of initially calculating the ratio of band intensity of each pre-rRNA species in the TAP-purified fractions to that for the same RNA species in total RNA fractions and then dividing this ratio by the ratio obtained for 25 S rRNA as described in Harnpicharnchai et al. (10). It should be noted that because the 35 S and 25.5 S pre-rRNAs were assayed by primer extension whereas other pre-rRNA species were assayed by Northern analysis, their relative enrichments cannot be directly compared. C, Hrr25p and Tif6p coimmunoprecipitate with the 66 S preribosomal particles. Cell-free extracts prepared from W303α (lane a), KSY607 expressing HA-tagged Tif6p (lane b), and PRY101 (lane c), expressing both HA-Tif6p and Myc-Hrr25p, were immunoprecipitated with anti-HA antibodies under conditions of coimmunoprecipitation as described under “Experimental Procedures.” The washed immunocomplexes (Anti-HA CoIP) as well as aliquots of each cell extract (Total extract), before immunoprecipitation, were analyzed by Western blotting using anti-HA antibodies to detect HA-tagged Tif6p (lower panel) or anti-Myc antibodies to detect Myc-tagged Hrr25p (upper panel). D, extracts from PRY101 and W303α cells were immunoprecipitated under conditions of coimmunoprecipitation with either anti-Myc antibodies (lanes a and b) to precipitate Myc-tagged Hrr25p-containing complexes or with anti-HA antibodies (lanes c and d) to precipitate HA-Tif6p-containing complexes. Each RNA sample, isolated from the washed beads by phenol/chloroform extraction, was then analyzed by Northern blotting using an appropriate DNA probe that specifically detects 27 SA+B pre-rRNAs (Probe 6) or 20 S pre-rRNA (Probe 3).