5178 Nucleic Acids Research, 2010, Vol. 38, No. 15 pathway and consistently with pioneer work by sucrose gradient analyses (26), 90S pre-ribosomal particles, nuclear and cytoplasmic 43S pre-ribosomal particles and early, medium, late and cytoplasmic pre-60S r-particles (15,16,27) (for an outline, see Supplementary Figure S2A). Apparently, the complexity of the diﬀerent pre-ribosomal particles decreases during their maturation to r-subunits while concomitant structural rearrangements allow the stable incorporation of all r-proteins (15,16,28). Insights into the approximate timing of association and dissociation of some of the protein ribosome biogenesis factors have been obtained by studying the composition of distinct puriﬁed pre-60S complexes from wild-type (15,17) and mutant strains blocked at early, medium or late nuclear steps of ribosome maturation [e.g. see (29,30) and references therein]. It has also long been clear that both the pre-rRNAs (transcribed spacers and mature rRNA sequences) and r-proteins are essential for the pre-rRNA processing events and hence for eukaryotic ribosome synthesis (31–33). A systematic analysis of the role of yeast 40S r-proteins in maturation and transport of 43S pre-ribosomal particles has been performed (34). Recently, an equivalent approach has allowed the analyses of the role of 26 essential yeast 60S r-proteins (35). Before this analysis, the contribution to ribosome biogenesis of only few 60S r-proteins has been studied in detail (36–41). Moreover, the course of the assembly of the r-proteins has not been reported with suﬃcient precision, mainly due to the fact that r-proteins are common con- taminants in puriﬁed complexes, therefore it has been dif- ﬁcult to distinguish between unspeciﬁc and speciﬁc association of r-proteins to pre-ribosomal particles (42,43). Low resolution pictures of early and late r-protein assembly has been obtained by monitoring the kinetics of in vivo incorporation of labelled r-proteins into pre-ribosomal particles and cytoplasmic ribosomes in both yeast and HeLa cells (44,45). More recent investigations have analysed the incorporation of tagged yeast 40S r-proteins into 90S and 43S pre-ribosomal particles (46), which have allowed the identiﬁcation of some principles governing the assembly of the 40S r-subunits and the es- tablishment of a certain parallelism between the in vivo assembly of 40S r-subunits and the in vitro reconstitution data of bacterial 30S r-subunits (46). Unfortunately, an equivalent analysis of the incorporation of yeast 60S r-proteins into pre-ribosomal particles is still missing. We are interested in understanding the contribution of speciﬁc 60S r-proteins to ribosome biogenesis (38,41). In this study, we have undertaken the functional analysis of yeast L35 in ribosome synthesis. Eukaryotic L35 shares a notable sequence identity with archaeal and eubacterial L29, although L35 carries a speciﬁc C-terminal extension (Ribosomal Protein Gene Database, http://ribosome.med .miyazaki-u.ac.jp/ and Supplementary Figure S3). L35 is located close to L25 and L26, thus ﬂanking the nascent peptide exit tunnel (Supplementary Figure S4) in a similar fashion as their respective archaeal and eubacterial L29, L23 and L24 counterparts (47). L35 together with L25 may function as a general docking site for nascent poly- peptide chain-associated factors, such as N-terminal processing and modiﬁcation enzymes, chaperones, the signal recognition particle (SRP) and the Sec61/translocon complex (48,49). Regarding ribosome synthesis, it has been suggested by polysome proﬁle analyses that L35 is required for 60S r-subunit accumulation (50). Our results show that L35 is indeed required for the normal accumu- lation of 60S r-subunit since defective production of L35 causes defects in pre-rRNA processing of 27SA3 and 27SBL/S and in intra-nuclear transport and nuclear export of pre-60S r-particles. Moreover, we show that L35 assembles in the nucle(ol)us and stably interacts with 27S pre-rRNAs, therefore suggesting that it is added early in the assembly pathway. It has been sug- gested that zebraﬁsh RPL35, as many other r-protein genes, acts as an haploinsuﬃcient tumor suppressor by an as yet unknown mechanism (51). In this study, we also show that depletion of L35 leads to a mild delay in the G1 phase of the cell cycle. MATERIALS AND METHODS Strains and microbiological methods The yeast strains used in this study, which are derivatives of strain BY4741 (52), are listed in Supplementary Table S1. Strains RBY138 and RBY139 are haploid segregants of Y23889 and Y23834 (Euroscarf), respective- ly. Growth and handling of yeast and standard media were according to established procedures (53,54). Antibiotic-containing plates were prepared by adding the drugs from stock solutions into 2% rich glucose medium (YPD) before pouring the plates. Tetrad dissections were performed using a Singer MS micromanipulator. Escherichia coli DH5a strain was used for common cloning and propagation of plasmids (55). Plasmids The relevant information on the construction of the dif- ferent plasmids used in this study can be found in the Supplementary Data. The sequences of oligonucleotides used for the construction of plasmids are listed in Supplementary Table S2. Other plasmids used are described throughout the text. All plasmids used in this study are listed in Supplementary Table S3. Construction of a GAL::RPL35 allele and in vivo depletion of L35 RBY138 [YCplac33-RPL35A] was crossed to RBY168 [YCplac111], the resulting diploid was sporulated and tetrads were dissected. G418-resistant spore clones from at least three complete non-parental ditypes were trans- formed with pAS24-RPL35A, which allows expression of N-terminally HA-tagged L35 under the control of a GAL promoter. Transformants were restreaked on synthetic 2% galactose (SGal)-Leu plates and subjected to plasmid shuﬄing on 5-FOA-containing SGal plates at 30 C. One of these clones, RBY175 [pAS24-RPL35A], also named the GAL::RPL35 strain, was used for further work.