286 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 4. 30 extremity of the 21S-C pre-rRNA. (A) Five speciﬁc probes (ITS1-160, ITS1-605, ITS1-640, ITS1-721 and ITS1-779, numbers correspond to nucleotidic position within the ITS1) were designed to delineate, by northern blot, the 30-end of the 21S-C intermediate seen in the absence of bystin. The 21S-C RNA is detected with probe ITS1-605, but not with probes hybridizing downstream in the ITS-1 (see position of the probes in D). (B) Schematic representation of the 21S and 21S-C pre-rRNA species and position of the probes. (C) The ITS1-721 and ITS2 probe do not reveal any large species that would be complementary to the 21S-C pre-rRNA, but show disappearance of the 36S pre-rRNA upon bystin deletion. (D) Formation of the 36S pre-rRNA. (E) 30 RACE analysis with primer ITS1-583 shows accumulation of species ending around position 635 and 650 of the ITS1 in bystin-depleted cells. These species are also found in control cells. However, no pre-rRNA migrating above the 32S RNA was revealed with the ITS1-721 or the ITS2 probes in bystin-depleted cells (Figure 4C). We observed with these probes the disappearance of the 36S pre-rRNA (Figure 4C), a rare precursor that forms upon direct cleavage of early precursors at site E (Figure 4D), which conﬁrms that bystin depletion blocks cleavage at site E. To further delineate the 30-end of the 21S-C pre-RNA, we performed 30 RACE experiments with an oligonucleo- tide spanning nucleotides 582–599 in the ITS1. First, an anchor oligonucleotide was ligated into the 30-termini of RNAs isolated from whole-cell extracts in order to perform reverse transcription. RNAs with their 30-end between nucleotide 600 and the anchor were then ampliﬁed by PCR, subcloned and sequenced. As shown in Figure 4E, we found several RNAs ending between positions 620 and 650, both in control and in bystin-deﬁcient cells. RNAs from bystin-depleted cells were enriched in species ending around positions 635 and 650. These species were also found in wild-type cells and the heterogeneity of the 30 extremities is consistent with the often-smeary appearance of the band found below the 21S pre-rRNA on northern blots (Figure 3B). This suggests that the 21S pre-rRNA is processed by a 30–50 exonuclease after cleavage at site 2. In this hypothesis, the 21S-C could correspond either to the pause or the stalling of this exonuclease, or to the end product of this exonucleolytic processing. Thus, these data show that bystin participates in efﬁ- cient cleavage at site 2 and is strictly required for further processing of the 21S pre-rRNA. In addition, they uncover a new processing mechanism in the ITS1. hTsr1 knockdown delays pre-40S particle nuclear export In contrast to bystin, hTsr1 knockdown did not affect the levels of the 18S rRNA precursors when compared with control cells (Figure 3B). Formation of the large subunit rRNAs was not affected either, as seen on the same northern blot revealed with ITS2 probes. However, in the absence of hTsr1, the 18S/28S rRNA ratio strongly decreased, conﬁrming the impact of hTsr1 depletion on 40S ribosomal subunit formation observed by sucrose-gradient analyses. These data indicate that hTsr1 performs a late function in 18S rRNA maturation and that hTsr1-deﬁcient pre-40S particles are rapidly turned over. We next analyzed the impact of hTsr1 and bystin knockdown on the intracellular fate of pre-ribosomes by FISH, using probes complementary to transcribed spacers 50-ETS/ETS1, ITS1 and ITS2 (Figure 5). We included in