5182 Nucleic Acids Research, 2010, Vol. 38, No. 15 from liquid YPGal medium to YPD medium, the growth rate rapidly decrease to a doubling time of >10 h after 12 h in YPD, as compared with the 1.5–2 h for the isogenic wild-type control strain (data not shown). Western blot analysis revealed a marked reduction of L35 in the GAL::RPL35 cells that coincided with the decrease in the growth rate in YPD medium. Interestingly, L1 is also reduced but to a lesser extent and S8 levels remained practically unaﬀected during the time-course of the shift (Figure 2B). To examine the consequences of L35 depletion in ribosome biogenesis, we ﬁrst analysed polysome proﬁles from cell extracts of the GAL::RPL35 strain grown in YPGal or after shifting to YPD. As shown in Figure 2C, this strain showed normal polysome proﬁles when grown in YPGal, including normal levels of free 40S and 60S r-subunit, 80S and polysomes. However, when shifted for only 6 h to YPD, it showed a clear decrease in the levels of free 60S versus free 40S r-subunits, a strong decrease in the 80S peak and polysome and the appearance of large half-mer poly- somes. These features were still more pronounced upon a 12 h shift to YPD (Figure 2C). Wild-type cells showed no alteration in the polysome proﬁle when transferred to YPD (data not shown). Consistently, quantiﬁcation of total r-subunits by low Mg2+ sucrose gradients showed that L35 depletion leads to a 40% reduction of the 60S to 40S r-subunit ratio compared to the wild-type control after 12 h of depletion (data not shown). Altogether, these results indicate that depletion of L35 leads to a deﬁcit in 60S r-subunits relative to 40S r-subunits. Depletion of L35 delays rRNA processing at ITS2 To determine whether L35 is required for pre-rRNA pro- cessing, we analysed the eﬀects of L35 depletion on the kinetics of rRNA production by [5,6-3H]-uracil pulse-chase labelling experiments. Both the wild-type and GAL::RPL35 strains were ﬁrst transformed with an empty YCplac33 plasmid (CEN, URA3, see ‘Materials and Methods’ section) to make them prototrophic for uracil. Then, they were pre-grown in liquid SGal-Ura medium and ﬁnally shifted to liquid SD-Ura medium for 8 h. At this time point, the GAL::RPL35 strain was doubling every ca. 8–9 h compared with ca. 2 h for the wild-type strain. The cells were pulse-labelled for 2 min, then chased for 5, 15, 30 and 60 min with a large excess of non-radioactive uracil. Total RNA was extracted from each sample and analysed by agarose and acrylamide gel electrophoresis, followed by transfer to nylon membranes and ﬂuorography. In wild-type cells, the 35S precursor was converted rapidly into 32S and then into 27S and 20S species, which were further processed into mature 25S and 18S rRNA, respect- ively. After 15 min of chase, almost all label was in the mature rRNAs. In contrast, in the L35-depleted strain, synthesis of 25S and 5.8S rRNAs was inhibited relative to synthesis of 18S and 5S rRNAs (Figure 3A and B). Processing of 35S pre-rRNA was delayed since the 35S pre-rRNA could be detected after the pulse and the early chase time points. Consistently, traces of the aberrant 23S species appeared (Figure 3A). Processing of 27SB pre-rRNAs was also slowed since these pre-rRNAs persisted even after the 60 min of chase; as a consequence no labelled 25S and 5.8S rRNA were detected (Figure 3A and B). Pulse-chase analyses were also performed with the Drpl35A and Drpl35B strains. These revealed that the Drpl35A mutation led to similar, albeit milder, eﬀects as those observed upon L35 depletion, while the Drpl35B mutant only showed a slight delay of processing of both 35S and 27SB pre-rRNAs (data not shown). Thus, the deﬁcit in 60S r-subunits upon mutation in or depletion Figure 3. Depletion of L35 impairs 27S pre-rRNA processing. (A) Wild-type control strain BY4741 (Wild-type) and strain RBY175 (GAL::RPL35) were transformed with YCplac33 and then grown at 30 C in SGal-Ura and shifted for 8 h to SD-Ura. Cells were pulse-labelled with [5,6-3H]uracil for 2 min followed by a chase with a large excess of unlabelled uracil for the times indicated. Total RNA was extracted and 20 000 cpm was loaded and separated on (A) a 1.2% agarose-6% formaldehyde gel or (B) a 7% polyacrylamide-8M urea, transferred to nylon membranes and visualized by ﬂuorography. The positions of the diﬀerent pre-rRNAs and mature rRNA are indicated.