5186 Nucleic Acids Research, 2010, Vol. 38, No. 15 Figure 7. L35B associates with all pre-60S ribosomal particles. L35B-eGFP was aﬃnity puriﬁed from total cellular extracts of Drpl35B cells expressing L35B-eGFP with GFP-Trap _A beads as indicated in ‘Materials and Methods’ section. Wild-type cells, which express untagged L35B, serves as a negative control. RNA was ex- tracted from the pellets obtained after puriﬁcation (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for puriﬁcation (lanes T) and subjected to northern analysis of pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Figure S1A. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to as the percentage of each RNA recovered after puriﬁcation. 2C peak was more pronounced after a 12-h shift to YPD, which led to an increase in the 1C/2C area peak from 0.7 in YPGal to 1.4 after the 12 h-shift to YPD (Figure 8A). Microscopy inspection of DAPI-stained cells from the wild-type grown either in YPGal or shifted to YPD revealed normal cellular morphology and a mixture of unbudded cells ( 43% from 120 examined cells) and cells with variably sized buds (Figure 8B). Similar results were obtained for the GAL::RPL35 cells grown in YPGal medium. However, when the GAL::RPL35 cells were transferred to YPD medium, a progressive increase in the percentage of unbudded cells was observed, and after 12 h in YPD, about 65% cells from ca. 200 examined cells were small and unbudded (Figure 8B). Similar FACS sorting and microscopy analyses were also performed with the Drpl35A and Drpl35B strains. These revealed similar although slighter phenotypes than those of L35 depleted cells for only the Drpl35A mutant (data not shown). We conclude that the G1 phase of cell cycle is mildly delayed upon decreased production of L35. DISCUSSION In this work, we have addressed the role of yeast L35 r-protein in ribosome biogenesis. L35 is an evolutionarily conserved protein in eukaryotes; moreover, a notable sequence homology exists between the N-terminal and central sequences of L35 and the eubacterial and archaeal L29 (Supplementary Figure S3). According to the X-ray crystals of 50S r-subunits, prokaryotic L29 is a globular protein made up of helical bundle domain [(47); Supplementary Figure S4]. The analysis of the models derived from the cryo-electron microscopy data of the yeast and dog 60S r-subunits suggests that at least the N-terminal and central parts of L35 are structurally similar to prokaryotic L29 (3,4,47); however, the distinct C-terminal extension of L35 can not been modelled. In addition, prokaryotic L29 and eukaryotic L35 have been mapped very close to prokaryotic L23 and L24 and eu- karyotic L25 and L26, respectively, surrounding the nascent polypeptide exit tunnel ((3,4,47); Supplementary Figure S4). Due to this fact, L25 and L35 have been shown to be important for the contact of the ribosome with ribosome-associated enzymes, chaperones and complexes such as SRP or the Sec61/translocon (48,49). Finally, the 60S r-subunit models also reveal that L35 might bind mature 5.8S rRNA at equivalent positions to those archaeal L29 interacts in mature 23S rRNA within domain I [(47); Supplementary Figure S6]. Accordingly, it has been demonstrated that rat liver L35 binds in vitro to yeast 5.8S rRNA (82). Unfortunately, despite all this knowledge, there is almost no information available on the role of L35 or its prokaryotic L29 counterpart in ribosome biogenesis, excluding some polysome proﬁles obtained for the Drpl35A and Drpl35B strains (50). Herewith, we have analysed the consequences for ribosome biogenesis of either the deletion of RPL35A and RPL35B or the deple- tion of L35. Our results show that deletion of RPL35A leads to a severe impairment of growth while deletion of RPL35B has almost no eﬀect on growth (Figure 1). This situation is similar to that found for other many yeast