Esf2p, a U3-Associated Factor Required for Small-Subunit Processome Assembly and Compaction

ABSTRACT Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5′ external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5′ ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

[1]  S. Baserga,et al.  Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae. , 2004, Molecular cell.

[2]  J. Gallagher,et al.  The Small-Subunit Processome Is a Ribosome Assembly Intermediate , 2004, Eukaryotic Cell.

[3]  S. Baserga,et al.  The small subunit processome is required for cell cycle progression at G1. , 2004, Molecular biology of the cell.

[4]  J. Gallagher,et al.  RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components. , 2004, Genes & development.

[5]  X. Bustelo,et al.  Functional Characterization of Pwp2, a WD Family Protein Essential for the Assembly of the 90 S Pre-ribosomal Particle*♦ , 2004, Journal of Biological Chemistry.

[6]  M. Thiry,et al.  The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology , 2004, Molecular and Cellular Biology.

[7]  T. Hughes,et al.  Exploration of Essential Gene Functions via Titratable Promoter Alleles , 2004, Cell.

[8]  K. Thorn,et al.  Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae , 2004, Yeast.

[9]  T. Hughes,et al.  The Specificities of Four Yeast Dihydrouridine Synthases for Cytoplasmic tRNAs*[boxs] , 2004, Journal of Biological Chemistry.

[10]  D. Lafontaine,et al.  Dim2p, a KH-domain protein required for small ribosomal subunit synthesis. , 2004, RNA.

[11]  T. Hughes,et al.  ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae. , 2004, Nucleic acids research.

[12]  T. Hughes,et al.  High-definition macromolecular composition of yeast RNA-processing complexes. , 2004, Molecular cell.

[13]  Brian E Snydsman,et al.  Assigning function to yeast proteins by integration of technologies. , 2003, Molecular cell.

[14]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[15]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[16]  S. Baserga,et al.  Probing the yeast proteome for RNA-processing factors , 2003, Genome Biology.

[17]  Micheline Fromont-Racine,et al.  Ribosome assembly in eukaryotes. , 2003, Gene.

[18]  P. Gleizes,et al.  Sequential Protein Association with Nascent 60S Ribosomal Particles , 2003, Molecular and Cellular Biology.

[19]  Brendan J. Frey,et al.  A Panoramic View of Yeast Noncoding RNA Processing , 2003, Cell.

[20]  G. Pruijn,et al.  The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes. , 2003, Nucleic acids research.

[21]  E. Petfalski,et al.  The path from nucleolar 90S to cytoplasmic 40S pre‐ribosomes , 2003, The EMBO journal.

[22]  M. Nomura,et al.  In Exponentially Growing Saccharomyces cerevisiae Cells, rRNA Synthesis Is Determined by the Summed RNA Polymerase I Loading Rate Rather than by the Number of Active Genes , 2003, Molecular and Cellular Biology.

[23]  David Tollervey,et al.  60S pre‐ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm , 2002, The EMBO journal.

[24]  A. Fatica,et al.  Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro , 2002, Molecular and Cellular Biology.

[25]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[26]  Bernhard Kuster,et al.  90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. , 2002, Molecular cell.

[27]  J. Shabanowitz,et al.  A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis , 2002, Nature.

[28]  T. Kiss.  Small Nucleolar RNAs An Abundant Group of Noncoding RNAs with Diverse Cellular Functions , 2002, Cell.

[29]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[30]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[31]  M. Mann,et al.  Directed Proteomic Analysis of the Human Nucleolus , 2002, Current Biology.

[32]  J. R. Warner,et al.  Nascent Ribosomes , 2001, Cell.

[33]  J. Shabanowitz,et al.  Composition and functional characterization of yeast 66S ribosome assembly intermediates. , 2001, Molecular cell.

[34]  W. Filipowicz,et al.  Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast. , 2001, RNA.

[35]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Christiane Branlant,et al.  A Common Core RNP Structure Shared between the Small Nucleoar Box C/D RNPs and the Spliceosomal U4 snRNP , 2000, Cell.

[37]  M. Caizergues-Ferrer,et al.  Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2. , 2000, RNA.

[38]  W. Filipowicz,et al.  Rcl1p, the yeast protein similar to the RNA 3′‐phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis , 2000, The EMBO journal.

[39]  Y. Masuho,et al.  A Novel TATA-Binding Protein-Binding Protein, ABT1, Activates Basal Transcription and Has a Yeast Homolog That Is Essential for Growth , 2000, Molecular and Cellular Biology.

[40]  Patrick Linder,et al.  Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[41]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[42]  Amos Bairoch,et al.  Recent improvements to the PROSITE database , 2004, Nucleic Acids Res..

[43]  Peer Bork,et al.  SMART 4.0: towards genomic data integration , 2004, Nucleic Acids Res..

[44]  D. Lafontaine,et al.  Eukaryotic Ribosome Synthesis , 2004 .

[45]  David Tollervey,et al.  Making ribosomes. , 2002, Current opinion in cell biology.

[46]  M. Caizergues-Ferrer,et al.  Rrp 8 p is a yeast nucleolar protein functionally linked to Gar 1 p and involved in pre-rRNA cleavage at site A 2 , 2000 .

[47]  D. Tollervey,et al.  Ribosome synthesis in Saccharomyces cerevisiae. , 1999, Annual review of genetics.

[48]  R. Planta,et al.  Ribosomal precursor particles from yeast. , 1975, Experimental cell research.