Nucleic Acids Research, 2010, Vol. 38, No. 15 5185 Figure 6. L35B assembles in the nucleus as inferred upon inhibition of export of 60S pre-ribosomal particles. (A) Localization of L25-eGFP and L35B-eGFP in a LMB sensitive strain. Either L25-eGFP or L35B-eGFP was expressed in AJY1539 cells, which harbours the LMB sensitive crm1 (T539C) allele. Cells were incubated for 3 h at 30 C in the absence (-LMB) or presence of 200 ng/ml LMB (+LMB). The GFP signal was inspected by ﬂuorescence microscopy. Arrows point to nuclear ﬂuorescence. (B) Localization of L25-eGFP and L35-eGFP upon induction of a nmd3 dominant negative allele. Drpl35B cells expressing L25-eGFP or L35B-eGFP were transformed with the pRS316-GAL-nmd3D100 plasmid and transformants were grown in the presence of raﬃnose (Raf, SRaf-Leu-Ura medium). Then, galactose was added to fully induce the Nmd3 100 protein. The GFP signal was inspected by ﬂuorescence microscopy at the time indicated. Arrows point to nuclear ﬂuorescence. apparent lack of stochiometry observed for the puriﬁca- tion eﬃciency of 25S versus 5.8S and 5S rRNAs is due to the use of diﬀerent gel matrices for their resolution. Moreover, mature 18S rRNA was also eﬃciently co-puriﬁed with L35B-eGFP. This must reﬂect the common unspeciﬁc association of 40S with 60S r-subunits in 80S couples in Mg2+ containing buﬀers or ribosomes engaged in translation. Interestingly, puriﬁca- tion of L35B-eGFP yielded all precursors (27S and 7S pre-rRNAs) of 25S and 5.8S (Figure 7). Signiﬁcantly, quantiﬁcation analysis indicated that all these pre-rRNAs are enriched to similar extents in the L35B-eGFP puriﬁed samples. In clear contrast, only back- ground levels were detected for 35S and 20S pre-rRNAs. All these results are speciﬁc since no RNAs were detected upon aﬃnity puriﬁcation from extracts of the untagged strain (Figure 7). We conclude that L35B does not noticeably associate with 90S pre-ribosomal particles but is stably assembled into very early pre-60S r-particles. Depletion of L35 leads to cell-cycle delay at the G1 phase Recent studies indicate that the cell cycle can be impaired at diﬀerent stages upon mutational inactivation or deple- tion of individual yeast ribosome synthesis factors and r-proteins [e.g. see (27,41,79–81)]. To study the contribu- tion of L35 to cell-cycle progression, we analysed the cell-cycle status and the cellular morphology of the GAL::RPL35 and a isogenic wild-type strain under both permissive and non-permissive conditions. First, asyn- chronous wild-type cells were grown in YPGal medium and shifted for up to 12 h to YPD medium and then the DNA content was analysed by FACS sorting. We detected two nearly equal peaks corresponding to cells with unreplicated (1C) and duplicated genomes (2C) (Figure 8A). A similar pattern was observed for the GAL::RPL35 cells grown in YPGal medium (Figure 8A). However, after a 3-h shift to YPD, the 1C peak of GAL::RPL35 cells was slightly higher than the one of 2C. Notably, the imbalance between the 1C and