288 Nucleic Acids Research, 2011, Vol. 39, No. 1 these experiments cells depleted of RPS15, a ribosomal protein required for nuclear export (15). With the 50ITS1 probe, cells treated with scrambled siRNAs displayed a strong nucleolar labeling characteristic of the early pre-rRNA species, as well as a cytoplasmic signal indicating the presence in this compartment of 18S-E pre-rRNA, the last precursor to the 18S rRNA (Figure 5A). The labeling level of the nucleoplasm was compara- tively weak, attesting rapid trafﬁcking of pre-ribosomal particles through this compartment. In bystin-depleted cells, cytoplasmic labeling decreased whereas nucleoli were conspicuously brighter than in control cells, suggest- ing accumulation of pre-40S particles in this domain. In addition, nucleoplasmic foci were observed in 50% of bystin-depleted cells, as previously reported after knockdown of RPS19 or RPS24 (44,45). Using probes complementary to the 50 ETS, no signiﬁcant differences were detected in bystin-depleted cells compared with control cells (Figure 5C): this suggests that transcription and early pre-rRNA maturation are not affected, which is in accordance with the northern blot results. In hTsr1-depleted cells, nucleolar labeling was also more intense. In addition, ﬂuorescence was detected in the whole nucleoplasm, similar to that observed in RPS15-depleted cells (Figure 5A), indicating that, in the absence of hTsr1, pre-ribosomal particles are retained in the nucleus. In parallel, the cytoplasm showed almost no ﬂuorescence. This change in pre-40S particle distribution was attested by quantiﬁcation of the ﬂuorescence signal (Figure 5B). Nucleoplasmic retention in cells depleted of hTsr1 was speciﬁc of the late pre-40S particles, as it was not observed with the probes complementary to the 50 ETS (Figure 5C). To strengthen these data, we performed subcellular fractionation followed by northern blot analysis (Figure 6A). Consistent with FISH, the nuclear fraction was enriched in 18S-E pre-rRNA upon hTsr1 knockdown (Figure 6B). In bystin-depleted cells, the nuclear fraction was enriched with 41S, 21S and 21S-C pre-rRNAs, whereas the level of 18S-E pre-rRNA decreased, both in the nuclear and in the cytoplasmic fractions. However, the nuclear and cytoplasmic distribution of the 18S-E pre-rRNA still produced in bystin-depleted cells did not change (Figure 6B). These data show that assembly of hTsr1 is required for normal trafﬁcking of pre-40S par- ticles from the nucleus to the cytoplasm. DISCUSSION The data presented here indicate that bystin and hTsr1 are required at distinct steps of the pathway: bystin is neces- sary for efﬁcient cleavage of the ITS1 at site 2 and for further processing of the pre-rRNA; hTsr1 is involved downstream in nuclear export of the pre-40S particles and potentially in ﬁnal cytoplasmic processing. Yeast Enp1p is a nuclear protein, concentrated in the nucleolus. This pre-ribosomal factor is required for the earliest cleavages at sites A0, A1 and A2 of the 35S pre-rRNA. Enp1p associates with pre-ribosomes from the 90S particle to the cytoplasmic pre-40S particles (17). Figure 6. Nuclear retention of pre-40S particle upon depletion of hTsr1. (A) Northern blot analyses of total (T), cytoplasmic (C) or nuclear (N) pre-rRNAs, extracted from cells transfected with bystin, tsr1 or rps15 siRNAs for 48 h. RNAs were revealed either with the 50ITS1 probe or a mixture of ITS2b and ITS2d/e probes. A scramble siRNA was used as a negative control. The cytoplasmic fraction shows no contamination with nuclear pre-rRNAs. (B) The amount of 18S-E pre-rRNA in the cytoplasmic and the nuclear fractions was quantiﬁed by phosphorimaging. The histograms show the percentage of 18S-E pre-rRNA found in both fractions in two independent experiments, as calculated from the balance of the whole fractionation procedure. Depletion of Enp1p affects cleavage of the ETS1 at sites A0 and A1 and blocks processing of the ITS1 at site A2. Alternative cleavage in the ITS1 at site A3 generates the 23S and 21S aberrant intermediates, whereas 20S pre-rRNA production is blocked (21). Here, we ﬁnd that the bystin/hEnp1 human protein is also implicated in pre-40S particle maturation. It is largely located in the nucleus and accumulates in the nucleolus, but it also accompanies the pre-40S particles to the cytosol. Our results conﬁrm previous studies on the intra- cellular localization of bystin, its presence in pre-40S par- ticles (26,40), and its involvement in ITS1 maturation (38,40). We extend these observations by showing that