284 Nucleic Acids Research, 2011, Vol. 39, No. 1 multi-labeling experiments, multiband dichroic mirrors (Chroma, Rockingham, VT, USA) were used to avoid pixel shift in the acquisition plane. Speciﬁc single-band emission ﬁlters were mounted on a motorized wheel (PI). Images were captured with the same exposure times for different samples. The gray levels were scaled within the same limits in order to allow sample comparison. Fluorescence quantiﬁcation of regions of interest was per- formed on 12-bit images using MetaMorph software. After background subtraction, the results were expressed as an amount of gray levels/pixel. Fluorescence was quantiﬁed in four identical regions of interest in the nu- cleoplasm and the cytoplasm in at least 24 cells per sample. RESULTS Human orthologs of Enp1p and Tsr1p Bystin presents 55% similarity and 36% identity with yeast Enp1p, whereas hTsr1 and Tsr1p share 51% simi- larity and 30% identity. Similarity between bystin and Enp1p reaches 75% (50% identity) in the C-terminal core domain which deﬁnes the speciﬁc ‘bystin domain’, whereas their N-termini are more divergent. The bystin domain of Enp1p has a truncated 50 extremity (Figure 2A). Two highly conserved domains are found in hTsr1 and Tsr1p: the DUF663 domain, whose function is unknown but which is also found in Bms1; and the AARP2CN domain, which is weakly similar to the GTP-binding domain of elongation factor TU. When expressed as GFP fusions, both proteins were mainly located in the nucleus and concentrated in the nu- cleolus, while the cytoplasm was faintly stained (Figure 2B). For bystin, this staining pattern was similar to immunodetection of the endogenous protein (data not shown) and conﬁrmed data from previous reports (26,38,40). After arrest of RNA polymerase I transcription and segregation of the nucleolar components by actinomycin D treatment (42), both GFP-bystin and GFP-hTsr1 were detected in the crescent-shape granular component, consistent with their putative association with pre-ribosomal particles in the nucleolus (Figure 2B). Bystin and hTsr1 are necessary for 40S ribosomal subunit formation Requirement of bystin or hTsr1 for ribosome synthesis was assessed by knocking-down expression of these proteins with siRNAs. In both cases, mRNA levels were reduced by 84–90% (data not shown). Ribosome produc- tion after bystin- or hTsr1 depletion was analyzed from cytoplasmic fractions prepared in the presence of cycloheximide and separated on sucrose density gradients (Figure 3A). Control cells electro-transformed with a scrambled siRNA displayed a typical proﬁle with peaks corresponding to the 40S and 60S subunits, 80S ribosomal particles and polysomes of increasing lengths. After knocking-down bystin, the levels of free 40S subunits and 80S particles were severely decreased, while 60S subunits accumulated. A similar phenotype was also observed upon depletion of hTsr1, although the decrease in 40S subunits was less pronounced. This imbalance in the levels of 40S and 60S subunits indicates a deﬁcit of 40S production. Consistently, the 18S/28S rRNA ratio was 30% lower in bystin- or hTsr1-deﬁcient cells than in control cells (see histogram in Figure 3C). Bystin depletion uncovers an additional step in ITS1 processing In order to determine the pre-rRNA processing steps of the 40S particle maturation pathway involving bystin and hTsr1, pre-rRNAs were analyzed by northern blot with probes hybridizing to the ITS1 or the ITS2 (Figures 3 and 4). In parallel, the intracellular fate of the pre-ribosomes was monitored by FISH with probes Figure 2. Structural domains and intracellular localization of bystin and hTsr1. (A) A search for speciﬁc protein domains was performed with InterproScan. Bystin and Enp1p present a ‘bystin domain’, characteristic of the bystin protein family. hTsr1 and Tsr1p have an AARP2CN domain, similar to the GTP-binding domain of the elongation factor TU, and a DUF663 domain of unknown function which is also found in Bms1. (B) Cells transfected with plasmids pGFP, pGFP-bystin or pGFP-hTsr1 were observed after chemical ﬁxation and nucleus counterstaining with Hoechst 33342. Treatment with 50 ng/ml actinomycin D for 3 h provokes nucleolar segregation. Boxes show individual nucleoli with a labeled crescent, the characteristic shape of the granular component in segregated nucleoli. Bar = 10 mm.