depletion of bystin leads to an accumulation of the 41S and 21S pre-rRNAs, which we interpret as a delay in ITS1 cleavage at site 2 and a blockade of ITS1 processing in the pre-40S particles. In addition, a short form of the 21S pre-rRNA accumulates. Given that this species is also detected at low levels in control cells, we think that it could be a short-lived intermediate between the 21S and 18S-E pre-rRNAs. Interestingly, a similar pre-rRNA is observed in human cells upon knockdown of ribosomal protein RPS19 (43), a protein whose depletion in yeast also results in cleavage at site A3 instead of A2 and strongly blocks ITS1 maturation in pre-40S particles. The presence of 21S-C pre-rRNA could indicate an alter- native endonucleolytic cleavage in the ITS1 preferentially used upon deﬁcient cleavage at site 2. However, we did not detect any cleavage product complementary to the 21S-C pre-rRNA by northern blot. In addition, the heterogeneity of the RNA 30 extremities found in this region by 30-RACE experiments, together with the fuzzy appearance of this band on northern blots, rather suggest that an exo- nuclease trims the 21S pre-rRNA after cleavage at site 2. We cannot exclude that this exonuclease is part of a deg- radation process triggered by the accumulation of 21S pre-rRNA in bystin-depleted cells. However, the presence in control cells of intermediates similar to those found upon bystin knockdown rather supports that this exonucleolytic processing is part of the 21S to 18S-E pre-rRNA conversion and constitutes an additional step in the maturation of the human pre-40S particles. Accumulation of 21S pre-rRNA as well as disappear- ance of 36S pre-rRNA upon bystin depletion indicate that bystin is required for cleavage at site E. Processing at site E also requires some RPS proteins, especially RPS18 and RPS19 (46). That RPS19 and bystin depletion yields similar phenotypes may indicate that RPS19 is required for recruitment of bystin to pre-40S particles, as observed in yeast (47). Interestingly, Enp1p and Rps19p in yeast are necessary for ITS1 cleavage at site A2. This draws an interesting parallel between yeast site A2 and human site E, although they are, respectively, positioned at 217 nt and 24 nt from the 18S rRNA 30-end. Similar to site A2, cleavage at site E seems to be dependent on processing of the 50-ETS, as seen upon depletion of some RPS proteins (45,46). Human site 2 in turn is independent of 50-ETS processing, like site A3 in yeast. The role of bystin in ITS1 processing and its association with pre-40S particles indicate that it is a true functional ortholog of yeast Enp1p. However, bystin does not com- plement a yeast enp1 mutant (21). This may be due to the strong divergence of the N-terminal domains of these proteins, which could be required for species-speciﬁc func- tions. In addition, we ﬁnd no evidence that bystin/hEnp1 is implicated in 50-ETS maturation, which may be a sig- niﬁcant difference in the functions of these proteins. Yeast Tsr1p localizes to the cytoplasm and the nucleus, including the nucleolus. It associates speciﬁcally with pre-40S particles, as indicated by its sole interaction with 20S pre-rRNA and pre-40S-speciﬁc factors (27,29). Tsr1p (Twenty-S rRNA) was named due to the large accumula- tion of 20S pre-rRNA resulting from depletion of this Nucleic Acids Research, 2011, Vol. 39, No. 1 289 factor (22,29); when detected by FISH, pre-40S particles are found to pile-up in the cytoplasm (29). The 40S/60S subunits imbalance detected here on sucrose gradients, conﬁrmed by the reduction of the 18S/28S ratio, argues for a major role of hTsr1 function in 40S subunit production. However, unlike in yeast, we observed no major accumulation of pre-rRNAs in the cytoplasm of HeLa cells treated with tsr1 siRNAs, but instead nucleoplasmic retention of pre-40S particles. This discrepancy may correspond to different coordination between pre-rRNA maturation and nuclear export of pre-40S particles in yeast and mammalian cells. Indeed, production of 18S rRNA requires two pre-rRNA process- ing steps in human pre-40S particles instead of one in yeast. Our results indicate that hTsr1 is not necessary for the ﬁrst step, i.e. conversion of 21S to 18S-E pre-rRNA. By analogy with yeast Tsr1p, hTsr1 may thus be involved in the ﬁnal cytoplasmic processing step. But prior to this second processing step, assembly of hTsr1 is required for efﬁcient nuclear export. Whether hTsr1 has a direct role in pre-ribosomal transport to the cytoplasm, for example, as an exportin adaptor, remains to be demonstrated. We found no leucine-rich nuclear export sequence in hTsr1 by computer search, unlike recently shown for hRio2 (26). This does not formally exclude the presence of such a sequence, given the weak consensus by which it is deﬁned. The absence in the mam- malian orthologs of Mex67p and Mtr2p of a loop critical for the function of these factors in yeast pre-60S particle nuclear export illustrates that pre-ribosomal transport has evolved between yeast and mammals (48). Alternatively, hTsr1 may be more indirectly required for correct assembly or folding of the pre-40S particles prior to nuclear export. The fact that 18S-E pre-rRNA does not accumulate in hTsr1-depleted cells suggests that hTsr1- deﬁcient pre-40S particles are very labile and more rapidly degraded in mammalian cells than in yeast. In conclusion, these data illustrate that, although the pre-40S particle-speciﬁc factors have clear functional homologies, maturation and nuclear export of these par- ticles are not governed by strictly similar mechanisms in yeast and human cells. We propose that this is linked to differences in ITS1 processing, which change the timing between the maturation of the particle and its competence for nuclear export. In addition, the potential additional pre-rRNA processing step revealed upon knockdown of bystin or RPS19 indicate that nuclear processing of the ITS1 in mammalian cells may be even more complex than currently envisioned. ACKNOWLEDGEMENTS The authors wish to thank Guillaume Stahl, Ce´ lia Plisson-Chastang and Milena Preti for scientiﬁc discus- sions, as well as Ulrike Kutay and S e´bastien Fribourg for sharing reagents. They are also indebted to Caroline Monod for proofreading the manuscript. C.C. designed and performed most of the experiments, M.-F.O’D. and V.C. and M.F. performed some of the experiments.