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J. Biol. Chem., Vol. 279, Issue 36, 37385-37397, September 3, 2004

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Functional Characterization of Pwp2, a WD Family Protein Essential for the Assembly of the 90 S Pre-ribosomal Particle^*

Mercedes Dosil and Xosé R. Bustelo

From the Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, University of Salamanca-CSIC, Campus Unamuno, E-37007 Salamanca, Spain

Here we report the functional characterization of Pwp2, an evolutionary conserved component of the 90 S pre-ribosome. Conditional depletion of the Pwp2 protein in yeast specifically impairs pre-rRNA proccessing at sites A₀, A₁, and A₂, leading to a strong decrease in 18 S rRNA and 40 S ribosomal subunit levels. Pre-ribosomal particle sedimentation analysis indicated that these defects are caused by a block in the formation of 90 S pre-ribosomes. We demonstrate that in Pwp2-depleted cells the U3 small nucleolar ribonucleoprotein is not able to interact with the 35 S pre-rRNA and accumulates as a free complex. Similarly, other 90 S particle components such as Imp3 and Imp4 do not associate with the pre-rRNA precursor in the absence of Pwp2. In addition, we have found that after blocking U3 ribonucleoprotein assembly, Pwp2 predominantly accumulates as a complex in association with five proteins: Dip2, Utp6, Utp13, Utp18, and Utp21. Immunoprecipitation and gradient sedimentation analysis revealed that this Pwp2 small subcomplex is capable of interacting directly with the 35 S pre-rRNA 5' end. Taken together, these results indicate that Pwp2 forms part of a stable particle subunit independent of the U3 small nucleolar ribonucleoprotein that is essential for the initial assembly steps of the 90 S pre-ribosome.

Received for publication, May 3, 2004 , and in revised form, June 23, 2004.

^* This work was supported by research grants from the Programa General del Conocimiento of the Spanish Ministry of Science and Technology (BMC2002-02992) (to M. D.), the Ministry of Education and Culture of the Autonomous Government of Castilla-León (SA040/02) (to M. D.), and by NCI, National Institutes of Health, Grant CA7373501 (to X. R. B.). The Centro de Investigación del Cáncer was supported by endowments from the Consejo Superior de Investigaciones Científicas, Castilla-León Autonomous Government, the Spanish National Network of Cancer Centers (C03/10, Carlos III Institute, Spanish Ministry of Health), and the Foundation for Cancer Research of Salamanca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

Scientist of the Ramón y Cajal Program (Spanish Ministry of Science and Technology) associated with the University of Salamanca. To whom correspondence should be addressed. Tel.: 34-923-294802; Fax: 34-923-294743; E-mail: mdosil{at}usal.es.

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