Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle.
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 A(0), A(1), and A(2), 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.
Dosil
Mercedes
M
Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, University of Salamanca-CSIC, Campus Unamuno, E-37007 Salamanca, Spain. mdosil@usal.es
Bustelo
Xosé R
XR
D001483:Base Sequence; D017931:DNA Primers; D006868:Hydrolysis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D017931:DNA Primers; D009687:Nuclear Proteins; C100850:PWP2 protein, S cerevisiae; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C000602833:Utp18 protein, S cerevisiae; C532747:Utp6 protein, S cerevisiae