280–291 Nucleic Acids Research, 2011, Vol. 39, No. 1 doi:10.1093/nar/gkq734 Published online 30 August 2010 Analysis of two human pre-ribosomal factors, bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals Coralie Carron1,2, Marie-Franc¸ oise O’Donohue1,2, Vale´ rie Choesmel1,2, Marle` ne Faubladier1,2 and Pierre-Emmanuel Gleizes1,2,* 1Universite´ de Toulouse, UPS, Laboratoire de Biologie Mole´ culaire Eucaryote and 2CNRS, LBME, F-31000 Toulouse, France Received July 2, 2010; Revised August 3, 2010; Accepted August 4, 2010 ABSTRACT Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin deple- tion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 pro- cessing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells. INTRODUCTION During the past 10 years, proteomic and genetic studies have allowed the identiﬁcation of approximately 190 proteins that play a role in eukaryotic ribosome biogen- esis, from the initial nucleolar steps to ﬁnal processing in the cytoplasm (1–6). These proteins, called pre-ribosomal factors, display different functions including endo- or exoribonucleases, RNA helicases, chaperones, NTPases or methyl transferases (2,5). They are implicated in ribosome biogenesis but do not form part of mature ribosomes. Early ribosomal proteins and pre-ribosomal factors assemble co-transcriptionally in the nucleolus with the neo-synthesized pre-ribosomal RNA (pre-rRNA), forming the so-called 90S pre-ribosomal particle (3,7). The large polycistronic rRNA precursor contains the future 18S, 5.8S and 28S mature ribosomal RNAs ﬂanked by external and internal transcribed spacers (ETSs and ITSs), which are progressively removed through endonucleolytic cleavages and exonucleolytic processing (Figure 1A) (8,9). Early processing steps include enzymatic modiﬁcations of nucleotides, by 20-O-ribose methylation or pseudo-uridylation (10,11), together with removal of the 50- and 30-ETS and endonucleolytic cleavage of the ITS1 (Figure 1B and C). This latter step splits the 90S particle into pre-40S and pre-60S particles, which then follow two separate pro- cessing pathways that involve distinct pre-ribosomal factors (4,12). Both particles are exported from the nucleus and their maturation is completed in the cyto- plasm (13–16). Association and dissociation of these numerous pre-ribosomal factors along the maturation pathway renders the composition of pre-ribosomal par- ticles highly dynamic. The exact activity or function of most of the pre-ribosomal factors remains unclear, even when the step at which they are implicated is known. The list of pre-ribosomal factors composing the pre-40S particles is much shorter than that for the large subunit (3,17–19). Following cleavage of the ITS1, the majority of the factors present in the 90S particle dissociate, although a few remain part of the pre-40S particle, including Enp1p, Dim1p, Rrp12p and Bms1p (3,17). Dim1p, a methyltransferase associated with both the 90S and the pre-40S particles, methylates two adjacent nucleotides in *To whom correspondence should be addressed. Tel: +33 561 335 927; Fax: +33 561 335 886 ; Email: gleizes@ibcg.biotoul.fr 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.