of L35 is a consequence of impaired 27SB pre-rRNA pro- cessing leading to a reduced synthesis of both the mature 25S and 5.8S rRNAs. Steady-state levels of pre- and mature rRNAs were also analysed by northern hybridization and primer exten- sion. For this, total RNA was extracted from the wild-type and deletion mutants grown in YPD and during a time-course of L35 depletion. As shown in Figure 4A, following deletion of RPL35A and L35 deple- tion, the 35S pre-rRNA and the aberrant 23S species accumulated while 32S, 27SA2 and 20S pre-rRNAs decreased. In agreement with the 27SB pre-rRNA persist- ence seen in pulse chase analyses, steady-state levels of 27SB pre-rRNAs accumulated, while those of 7S pre-rRNA, mature 25S rRNA and to a lesser extent 5.8S rRNA decreased. Some alterations were also detected in the accumulation of mature 18S and 5S rRNAs (Figure 4A and B). The ratio between the long and short forms of mature 5.8S rRNAs was unaltered, suggest- ing that the alternative 27SA pre-rRNA processing pathways (Supplementary Figure S1) that generate the two 5.8S rRNA species both are impaired. To conﬁrm this, pre-rRNA processing was also assessed by primer extension analysis using an ITS2 probe (Supplementary Nucleic Acids Research, 2010, Vol. 38, No. 15 5183 Figure S1). As shown in Figure 4C, and consistent with the northern hybridization data, a decrease in the primer extension stop at site A2 is seen in the Drpl35A and the L35-depleted strains. The 27SA3 pre-rRNA, which cannot be readily detected in northern analyses, was accumulated in both the Drpl35A and the L35-depleted strains, as shown by the stop at site A3. Moreover, as suspected, both 27SBL and 27SBS pre-rRNA were also accumulated upon the deletion of RPL35A and the depletion of L35. Finally, primer exten- sion analysis was also performed through site C2, and the results showed a reduction in the levels of the 25.5S pre-rRNA (data not shown). Together, these data indicate that L35 is required for proper ITS2 pre-rRNA processing, thus, depletion of L35 strongly impairs cleavage at site C2. In addition, deletion of RPL35A and depletion of L35 delay processing at sites A0–A2 and site A3. Depletion of L35 impairs export of pre-60S r-particles from the nucleus to the cytoplasm To determine whether mutation in or depletion of L35 impairs nuclear export of pre-60S r-particles, Figure 4. Deletion of RPL35A and depletion of L35 aﬀects the steady-state levels of pre-rRNA and mature rRNA species. Strains BY4741 (Wild-type), RBY138 (Drpl35A) and RBY139 (Drpl35B) were grown in YPD at 30 C and harvested. BY4741 (Wild-type) and RBY175 (GAL::RPL35) were grown in YPGal at 30 C, shifted to YPD and harvested at the indicated times. Total RNA was extracted of each sample. Equal amount of total RNA (5 mg) was subjected to northern hybridization or primer extension analysis. (A) Northern analysis of high-molecular-mass pre-rRNAs and mature rRNAs. (B) Northern analysis of low-molecular-mass pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Supplementary Figure S1A. (C) Primer extension analysis with probe g within ITS2 allows detection of 27SA2, 27SA3 and both 27SB pre-rRNAs. Signal intensities were measured by phosphorimager scanning; values (indicated below each lane) were normalized to those obtained for the wild-type control grown either in YPD or YPGal, arbitrarily set at 1.0.