3198 Nucleic Acids Research, 2006, Vol. 34, No. 10 subunit to the 50 end of mRNA during translation (33,34). Based on solution structure studies, it has been proposed that binding of eIF4G to the C-terminal domain of eIF4A enhances the enzymatic activities by stabilizing an orientation of the two globular helicase domains that supports the ‘active’ conformation of eIF4A, a model that has been referred to as the ‘soft clamp’ model (34). Thus, it is tempting to speculate that Esf2 regulates Dbp8 activity by inducing a conformation that switches Dbp8 into its active state. Although we have demonstrated ATPase activity for Dbp8, we have not been able to show ATP hydrolysis-dependent strand separation (also sometimes referred to as ‘RNA unwinding’). Using RNA substrates with 12 bp duplexes, we could frequently detect strand separation of substrates containing 30 overhangs, but not blunt or substrates with 50 overhangs (S. Granneman, C. Y. Lin and S. J. Baserga, unpublished data). Unfortunately, this activity was also regu- larly observed in the absence of ATP and with an ATPase defective Dbp8 mutant protein, suggesting that binding of Dbp8 to the substrate with the 30 overhang was sufﬁcient to separate the strands. Thus, we have been unable to unequi- vocally demonstrate energy dependent RNA unwinding or strand separation by Dbp8, and have therefore not been able to adequately test the role of Esf2 in this process. Previous experiments have shown that Dbp8 and Esf2 are protein components of the large RNP, the SSU processome [also called 90S pre-ribosome; (19,40)], and as such are complexed with 40+ proteins and one or more snoRNAs. Consistent with SSU processome function, Esf2 has been found to be associated with 50ETS rRNA fragments by immunoprecipitation (19). One intriguing possibility is that Esf2, as an RNA binding protein, acts to facilitate the inter- action of Dbp8 with its RNA or RNP target. Thus, binding of Dbp8 to Esf2, possibly through its C-terminal coiled-coil domain, may not only activate Dbp8, but could also serve to place it near its site of action. Of note is that other studied DExD/H box helicase co-factors that stimulate ATPase activity (eIF4B, eIF4G and RNase E) are also RNA binding proteins, indicating that this is likely to be a general theme among helicase co-factors. Several possible functions have been ascribed to the RNA helicases involved in ribosome biogenesis. Because many have been found associated with large pre-ribosomal com- plexes, it has been suggested that they facilitate the numerous RNP remodeling steps, including disrupting rRNA–rRNA or protein–rRNA interactions, necessary for the maturation of ribosomal subunits. They could also facilitate rRNA–rRNA interaction, as helicases have been found to have an annealing activity (3). Another hypothesized function is that they unwind snoRNAs from the pre-rRNA after the chemical modiﬁcation or cleavage steps have occurred. Consistent with this model, the DExD/H box RNA helicase Dbp4 has recently been shown to be required for the release of the U14 snoRNA from the pre-rRNA, although it remains unclear whether it is Dbp4 that unwinds the duplex (15). In all cases, the natural RNA or RNP substrates of the helicases involved in ribosome biogenesis have unfortunately remained elusive (11,12,15,41,42). It is probable that, like Dbp8, the seventeen other DExD/H box helicases required for ribosome bio- genesis carry out their function in rRNA unwinding, RNP remodeling or rRNA annealing in conjunction with a speciﬁc co-factor that stimulates their activity and perhaps directs them to their target substrate. ACKNOWLEDGEMENTS The authors are grateful to Wendy Bussen, Stephen van Komen, Margaret Macris, Michael Sehorn and Patrick Sung for help and advice on protein purifications and ATP hydrolysis assays and providing reagents and their facilities. The authors are grateful to Amanda Solem, Olga Federova and Anna Marie Pyle for their advice and for providing reagents and their facilities. This work was supported by Leslie H. Warner and Anna Fuller Postdoctoral Cancer Research fellowships (S.G.), Arnold and Mabel Beckman Foundation (C.Y.L.) and the National Institutes of Health to S.J.B. (GM52581). Funding to pay the Open Access publication charges for this article was provided by NIH GM52581. Conflict of interest statement. None declared. REFERENCES 1. Jankowsky,E., Gross,C.H., Shuman,S. and Pyle,A.M. (2000) The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature, 403, 447–451. 2. Jankowsky,E., Gross,C.H., Shuman,S. and Pyle,A.M. (2001) Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science, 291, 121–125. 3. Yang,Q. and Jankowsky,E. (2005) ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1. Biochemistry, 44, 13591–13601. 4. Fairman,M.E., Maroney,P.A., Wang,W., Bowers,H.A., Gollnick,P., Nilsen,T.W. and Jankowsky,E. (2004) Protein displacement by DExH/D ‘RNA helicases’ without duplex unwinding. Science, 304, 730–734. 5. Rocak,S. and Linder,P. (2004) DEAD-box proteins: the driving forces behind RNA metabolism. Nature Rev. Mol. Cell. Biol., 5, 232–241. 6. Cordin,O., Banroques,J., Tanner,N.K. and Linder,P. (2006) The DEAD-box protein family of RNA helicases. Gene, 367, 17–37. 7. Tanner,K.N., Cordin,O., Banroques,J., Doere,M. and Linder,P. (2003) The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol. Cell., 11, 127–138. 8. Cordin,O., Tanner,N.K., Doere,M., Linder,P. and Banroques,J. (2004) The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. EMBO J., 23, 2478–2487. 9. Silverman,E., Edwalds-Gilbert,G. and Lin,R.J. (2003) DExD/H-box proteins and their partners: helping RNA helicases unwind. Gene, 312, 1–16. 10. Raue,H.A. (2004) Pre-ribosomal RNA processing and assembly in Saccharomyces cerevisiae. In Olson,M.O. (ed.), The Nucleolus. Kluwer Academic/Plenum Publishers, NY, pp. 199–222. 11. Bernstein,K.A., Granneman,S., Lee,A.V., Manickam,S. and Baserga,S.J. (2006) Comprehensive mutational analysis of yeast DEXD/H box RNA helicases involved in large ribosomal subunit biogenesis. Mol. Cell. Biol., 26, 1195–1208. 12. Granneman,S., Bernstein,K.A., Bleichert,F. and Baserga,S.J. (2006) Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis. Mol. Cell. Biol., 26, 1183–1194. 13. Daugeron,M.C. and Linder,P. (2001) Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis. Nucleic Acids Res., 29, 1144–1155. 14. Kikuma,T., Ohtsu,M., Utsugi,T., Koga,S., Okuhara,K., Eki,T., Fujimori,F. and Murakami,Y. (2004) Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity. J. Biol. Chem., 279, 20692–20698. 15. Kos,M. and Tollervey,D. (2005) The putative RNA helicase Dbp4p is required for release of the U14 snoRNA from preribosomes in Saccharomyces cerevisiae. Mol. Cell., 20, 53–64.