Nucleic Acids Research, 2010, Vol. 38, No. 15 5187 Figure 8. Depletion of L35 leads to a mild delay of the cell cycle at the G1 phase. (A) FACS analysis of unsynchronized cells from the BY4741 (Wild-type) or the RBY175 (GAL::RPL35) strains. Cells were grown in YPGal (Gal) or shifted for up to 12 h to YPD (Glc) at 30 C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively. Numbers refers to the 1C/2C area peak ratios (B) Cell morphology of the wild-type and GAL::RPL35 cells. Cells were stained with DAPI for localization of nuclei and then visualized by ﬂuorescence and visible phase contrast microscopy. Only merged images are shown. Numbers refer to mean percentages of unbudded cells after three independent experiments. r-proteins encoded by duplicated genes, among them L15 (83), L11 (84) and L2 (85). In agreement with the previous report (50), polysome analysis reveals a more drastic 60S r-subunit deﬁcit for the Drpl35A than for the Drpl35B strain. Consistent with this, western blot analysis suggests that RPL35A plays a more important role in the production of L35 than RPL35B (Figure 1). As expected, depletion of L35 leads to an even stronger deﬁcit in 60S r-subunits (Figure 2). Analysis of pre-rRNA processing by pulse-chase labeling and northern blotting analysis clearly indicates that the 60S r-subunit deﬁcit exhibited by the rpl35 mutants is due to a strong inhibition of processing of the 27SB pre-rRNAs, which leads to reduced formation of 7S and 25.5S pre-rRNAs and thus of mature 5.8S and 25S rRNAs, re- spectively (Figures 3, 4 and data not shown). Pulse-chase and northern blot analysis also indicate that deletion of RPL35A and depletion of L35 cause a decrease in the eﬃciency of processing at the early cleavage sites A0, A1 and A2, thereby aﬀecting the levels of mature 18S rRNA and its 20S precursor and leading to the appearance and accumulation of the aberrant 23S pre-rRNA (Figures 3 and 4). This type of defect in 18S rRNA synthesis, which has been extensively reported, is a feature of many mutants in genes encoding 60S r-subunit synthesis factors or 60S r-proteins (5,11,41,86). Northern and primer extension analysis also show an accumulation of 27SA3 pre-rRNA in the Drpl35A and the L35-depleted strain, however, the ratio of the mature 5.8SL and 5.8SS rRNAs is not altered as observed upon mutation or de- pletion of RNase MRP, which is the endonuclease respon- sible for the cleavage at site A3 (87,88). These pre-rRNA processing defects, at least the accumulation of 27SB pre-rRNAs, closely resemble those described upon mutation in or depletion of some 60S r-subunit biogenesis factors, such as Dbp10 (89), Mak11 (90), Nip7 (91), Nog1 (30), Nop2 (92), Nop3 (93), Rlp24 (30), Spb1 (94) and Spb4 (95). The levels of 27SA3 pre-rRNAs were checked only upon depletion of Spb1 and Spb4 and in both cases, similarly as upon L35 depletion, there was a clear increase of this precursor, especially at early depletion time points (94,95). Interestingly, depletion of r-protein L25 as well as mutations in either the L25-binding site within domain III of 25S rRNA or in L25, which abolish its association with this rRNA site also resulted in a notable inhibition of 27SB pre-rRNA processing (40,96,97). Peculis and co-workers have also demonstrated that formation of the so-called ITS2 proximal stem in which the 30-end of 5.8S rRNA base-pairs with the 50-end of 25S rRNA is a prerequisite for eﬃcient 27SB pre-rRNA processing (32,98,99). All these results and observations strongly suggest that assembly of L25 and/or L35 may be required to achieve the proper structural conformation in the ITS2 region of the 27SB-preRNAs for eﬃcient cleavage at site C2. Nevertheless, it is unlikely that L35, which lacks known nuclease motifs, represents the still unknown endonuclease responsible for this cleavage. Further experiments are required to identify this endo- nuclease and test its link with L25 and L35 and factors such as Spb4 that are required for 27SB pre-rRNA pro- cessing; one possibility is that interaction of any of these factors with 27SB pre-rRNA containing pre-60S r-particles could be dependent on previous association of L25 and L35 r-proteins. Recently, it has been proposed that 20S to 18S pre-rRNA processing by endonucleolytic cleavage at site D is promoted by a structural rearrange- ment of cytoplasmic 43S pre-ribosomal particles (100). This requires, in addition to the endonuclease Nob1 and