We present several lines of evidence that Esf1p is involved directly in 18S rRNA biogenesis. First, depletion of Esf1p led to a virtually complete loss of 20S and 27SA2
precursors, suggesting that the cleavage at site A2
was blocked in the mutant (Fig. ). In addition, the aberrant 23S RNA species and 35S precursor accumulated in the mutant, indicating inhibition of cleavage events at A0
). Although 18S rRNA biogenesis was affected in the mutant, 25S rRNA levels remained largely normal, suggesting that the 5.8S and 25S rRNAs are formed normally following cleavage at A3
(Fig. ). Studies of kinetics of rRNA formation in the mutant also suggested that Esf1p mainly affected the biogenesis of 18S rRNA (Fig. ).
Among the proteins that co-purified with Esf1p, several are involved in 18S rRNA biogenesis. The nucleolar protein Nop1p is a component of the SSU processome complex (4
). Nop1p is also a core component of all Box C/D snoRNPs (2
), which include both U3 and U14 snoRNPs. Krr1p is required for 40S ribosome biogenesis (24
) and subsequent export to the cytoplasm (5
) and was found in the 90S pre-ribosome (5
), although in our hands it does not co-purify with U3 or U14 RNAs, consistent with previous results (6
) (Fig. ). Nsr1p, a homolog of mammalian nucleolin (25
), contains two RRMs (RNA recognition motifs) and a glycine/arginine-rich (GAR) domain. It is required for 18S rRNA synthesis (25
). Utp22p is also involved in maturation of pre-18S rRNA (5
) and has been described as a component of the SSU processome (27
), although we have previously identified Utp22p in a distinct processome ‘sub-complex’ containing Rrp7p and yeast casein kinase (5
). Aside from Utp22p and Nop1p, we did not detect association of Esf1p with any of the components of the SSU processome, indicating that it is not a core component of this large RNP.
Hazbun and colleagues previously identified an Esf1p-associated complex which differs dramatically from ours, containing Bfr2p, Enp2p, Hca4p, Lcp5p, Nop58p and Utp9p (21
). Among these proteins, Hca4p, Lcp5p, Nop58p and Utp9p are involved in 18S rRNA synthesis (2
). Hca4p is a putative DEAD box RNA helicase and over-expression of HCA4
can suppress a U14 mutant (28
). Lcp5p was found to be associated with U3 snoRNA (29
) and Nop58p and Utp9p were found in both the SSU processome and 90S pre-ribosome (4
), although Lcp5p was not found in the SSU processome (4
). Bfr2p was also found in the 90S pre-ribosome (6
). Hence, Hazbun and colleagues’ data also support the general conclusion that Esf1p is involved in small-subunit biogenesis, although there is a complete lack of overlap with the specific associations we observed.
We propose that Esf1p-containing complexes may be dynamic. Although Esf1p co-purifies with two individual components of the SSU processome (Utp22p and Nop1p) (4
) (Fig. ), as well as the U3 and U14 snoRNAs and the 5′-ETS of the rRNA (Fig. ), all of the associations appear to be sub-stoichiometric, with the possible exception of the ribosomal proteins themselves. This is also clearly the case for the complex described in Hazbun et al
). Dynamic complexes might explain why different laboratories obtain quite different results from affinity purifications, since the associated proteins might depend upon the growth phase at harvest, the extraction procedure and the purification protocol used. The presence of Esf1p in multiple pre-ribosomal processing intermediates might also underlie its association with protein components of both the large and small ribosomal subunits. Analysis of mutants in the conserved features of Esf1p (Fig. ) might help resolve the biochemical role of Esf1p in 18S biogenesis: presumably, they represent discrete domains that mediate physical interactions and/or catalytic functions.