5188 Nucleic Acids Research, 2010, Vol. 38, No. 15 the RNA helicase Prp43 (100), the participation of S3 (28,34) and the C-terminal end of S14 (101) among other 40S r-proteins such as S0, S2, S10, S15, S20, S21, S26 and S28, which all are needed for eﬃcient 20S pre-rRNA processing to mature 18S (34,102). We wonder if a similar scenario would apply for the function of r-proteins L25 and L35, the C2-site endonucle- ase and the RNA helicases Spb4 and Dpb10. In addition to pre-rRNA processing defects, very rapidly upon L35 depletion and in Drpl35A cells, there is an accumulation of the L25-eGFP but not of the S2-eGFP reporter ﬁrst in the nucleolus and later in the whole nucleus (Figure 5). This result suggests that L25-GFP con- taining pre-60S r-particles are being retained in the nucle(ol)us due to impaired intra-nuclear and nucleo-cytoplasmic export. This phenotype has previously been observed in several mutants aﬀecting 60S r-subunit biogenesis [e.g. see (103) and references therein], including some mutants of 60S r-protein genes (38,41,62). As earlier discussed (103), this phenotype is likely not the conse- quence of a defect in the bona ﬁde export machinery but of a quality control system that retains defective pre-60S r-subunit particles. L25 has been suggested to assemble at an early stage of the 60S r-subunit maturation pathway (45), thus since the rpl35 mutants accumulate 27SB pre-rRNAs, the L25-eGFP ﬂuorescence signal observed at early time points of L35 depletion would mainly cor- respond to nucleolar, aberrant early E2 pre-60S r-particles. This is consistent with the fact that 27SB pre-rRNA pro- cessing at site C2 most likely occurs in the nucleolus (23,104). Export of pre-60S r-particles requires the partici- pation of several nuclear export receptors among them Arx1 (105,106). Results of Johnson0s laboratory strongly suggest that Arx1 binds in the vicinity of L25 and L35 at the exit tunnel of late pre-60S r-particles (107). This is consistent with the fact that Arx1 is structurally related to methionine aminopeptidases, which as mentioned above, interact with translating ribosomes via the outside part of the nascent peptide exit tunnel (48). Therefore, some of the accumulation of L25-eGFP in the nucleoplasm, seen in the L35-depleted strain at late time points, could reﬂect the loss of Arx1 binding to aberrant pre-60S r-particles. Indeed, our initial experi- ments show that the sedimentation behavior of Arx1 is altered upon L35-depletion. Thus, in the GAL::RPL35 strain in permissive conditions, Arx1 co-sediments with both free fractions and large complexes that could corres- pond to pre-60S r-particles in sucrose gradients. However, upon L35-depletion, Arx1 is largely absent from the 60S peak and is predominantly present at the top of the gradient. This suggests that Arx1 binding to pre-60S r-particles depends on the presence of L35 in these r-particles (R.B., unpublished results). In this work, we have also addressed the course of assembly of L35. No previous information was available for the yeast L35 r-protein even in the seminal work that addressed the kinetics of stable association of most r-proteins with pre-ribosomal particles (45). However, it could be demonstrated that human L35 r-protein binds to nucle(ol)ar pre-60S r-particles in HeLa cells (44). Consistently, it has also been shown in HeLa cells that L35 is imported to the nucleus due to the presence of a NLS within the C-terminal part of the protein (108). In this study, we present several lines of evidence that yeast L35 assembly occurs in the nucle(ol)us. Thus, we show that a functional GFP-tagged L35B protein accumulates in the nucleus upon inhibition of nucleo-cytoplasmic export of pre-60S r-particles (Figure 6), either by a LMB treatment or overexpression of the nmd3D100 allele. We have also examined the pre- and mature rRNAs with which aﬃnity-puriﬁed L35B-eGFP is associated (Figure 7). Our results strongly suggest that L35 is not present in 90S pre-ribosomal particles and it only stably binds to early pre-60S r-particles as soon as 27SA2 pre-rRNA is formed. These results are consistent with the fact that only few 60S r-subunit biogenesis factors and r-proteins have been detected in TAP-puriﬁcations of components of 90S pre-ribosomal particles (18). In E. coli, L29, the bacterial counterpart of yeast L35, is also an early assembling r-protein (109,110). Strikingly, binding of L29 to 23S rRNA in vitro is strongly dependent on L4, which lacks a known eukaryotic counterpart, and L23 and L24, which are the bacterial homologues of L25 and L26, respectively (Ribosomal Protein Gene Database, http://ribosome.med.miyazaki-u.ac.jp). As above men- tioned, yeast L25 and L26 are the closest neighbours of L35 (Supplementary Figure S4), thus, further experi- ments are required to determine whether in vivo as- sembly of yeast L25, L26 and L35 is interdependent. Supplementary Figure S2 summarizes the conclusions of this study concerning the L35 assembly in wild-type cells and the dynamics of 60S r-subunit maturation upon depletion of this 60S r-protein. While this manuscript was ﬁnalized, Po¨ ll et al. (35) reported a comparative analysis of the pre-rRNA process- ing and nucleo-cytoplasmic transport defects that arise upon depletion of 26 diﬀerent yeast 60S r-proteins, among them L35. Consistent with our data, it was shown that depletion of L35 strongly delays 27SB pre-rRNA processing into 7S and 25.5S pre-rRNAs and inhibits export of pre-60S r-particles. Interestingly, not only L35 and the expected L25 were found to be required for 27SB pre-rRNA processing, but also L9, L19, L23, L27 and L34 (35). Understanding how all these diﬀerent 60S r-proteins and L26, which was unfor- tunately not included in that study (35), do cooperate to promote eﬃcient cleavage at site C2 clearly requires further work. Finally, we report that L35 depletion results in small, unbudded cells that are mildly delayed in the G1 phase of the cell cycle (Figure 8). In yeast, cell-cycle defects have been previously described for mutants or strains depleted of r-proteins and biogenesis factors [see (27,41,81) and references therein]. Although, an impairment of diﬀerent cell-cycle stages could be observed, arrest in the G1 phase was the most common phenotype (41,80,81). This has been generally related to the intimate link between pro- gression through the G1 phase of the cell cycle and active protein synthesis, which is a prerequisite for acquiring the critical cell size and mass in order to pass the Start point (81,111). It has been suggested that zebraﬁsh RPL35, as many other r-protein genes, acts as an haploinsuﬃcient