Published online 14 April 2010 Nucleic Acids Research, 2010, Vol. 38, No. 15 5177–5192 doi:10.1093/nar/gkq260 Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae Reyes Babiano and Jesu´ s de la Cruz* Departamento de Gene´ tica, Universidad de Sevilla, Sevilla, Spain Received December 22, 2009; Revised March 19, 2010; Accepted March 29, 2010 ABSTRACT Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individ- ual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of riboso- mal protein L35 in ribosome biogenesis. In vivo de- pletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer poly- somes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C2. INTRODUCTION Ribosomes are highly complex ubiquitous ribonucleo- particles that are responsible for translating the mRNA into proteins (1). In all cells, ribosomes are composed of two ribosomal subunits (r-subunits). Recently, the struc- tural complexity of the eubacterial and archaeal ribo- somes has been obtained at the atomic resolution (2). Molecular models of eukaryotic ribosomes have also been documented for yeast and mammals. Although the resolution of these models is much lower than that from the crystals, its analysis reveals that eukaryotic ribosomes are more complex but display considerable structural simi- larity to their prokaryotic counterparts (3,4). Ribosome biogenesis is a complicated pathway that requires the coordinated assembly of the ribosomal RNAs (16S, 5S and 23S rRNAs in prokaryotes, 18S, 5S, 5.8S and 25S rRNAs in eukaryotes) with the r-proteins (about 50 diﬀerent proteins in prokaryotes and about 80 in eukaryotes) to yield the two r-subunits. In eukaryotes, this takes place largely within the nucleolus (5,6) although late steps occur in the nucleoplasm and even in the cyto- plasm (5,7,8). Ribosome biogenesis is evolutionary conserved throughout eukaryotes (9) but it has been studied most extensively in the yeast Saccharomyces cerevisiae (10,11). In the yeast nucleolus, a large precursor rRNA (pre-rRNA) encoding the mature 18S, 5.8S and 25S rRNAs is synthesized by RNA polymerase I whereas a pre-5S is independently transcribed by RNA polymerase III (12). The pre-rRNA processing pathway is now almost fully known and requires a series of sequen- tial endo- and exonucleolytic reactions that are mediated by endo- and exonucleases (for details, see Supplementary Figure S1). Pre-rRNA maturation (processing and rRNA modiﬁcation) and the assembly of pre-rRNA with r-proteins occur concomitantly, and the overall process requires about 200 protein factors and 80 small nucleolar RNAs (snoRNAs) (13–16). These factors likely assure the needed speed, accuracy and directionality of the ribosome assembly process (17). The consequences for ribosome synthesis due to loss-of-function mutations in most of these factors have been well described (5,10,11). Moreover, proteomic approaches have revealed several distinct, successive pre-ribosomal particles. The study of the RNA and protein composition of diﬀerent puriﬁed pre-ribosomal complexes is consistent with the presence of distinct intermediates that move from the nucleolus to the nucleoplasm and from there to the cytoplasm (15,18–25). These intermediates are termed, according to their position in the ribosome assembly *To whom correspondence should be addressed. Tel: +34 95 455 71 06; Fax: +34 95 455 71 04; Email: jdlcd@us.es The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.