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.

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A Direct Interaction between the Utp6 Half-a-Tetratricopeptide Repeat Domain and a Specific Peptide in Utp21 Is Essential for Efficient Pre-rRNA Processing

Molecular and Cellular Biology ◽

10.1128/mcb.00906-08 ◽

2008 ◽

Vol 28 (21) ◽

pp. 6547-6556 ◽

Cited By ~ 37

Author(s):

Erica A. Champion ◽

Bennett H. Lane ◽

Meredith E. Jackrel ◽

Lynne Regan ◽

Susan J. Baserga

Keyword(s):

Ribosome Biogenesis ◽

Direct Interaction ◽

Small Subunit ◽

Tetratricopeptide Repeat ◽

Peptide Ligand ◽

Rrna Processing ◽

Tetratricopeptide Repeat Domain ◽

Domain Peptide ◽

Ssu Processome ◽

Specific Peptide

ABSTRACT The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 μM.

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Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0249 ◽

2007 ◽

Vol 18 (2) ◽

pp. 394-403 ◽

Cited By ~ 29

Author(s):

K. Kopp ◽

J. Z. Gasiorowski ◽

D. Chen ◽

R. Gilmore ◽

J. T. Norton ◽

...

Keyword(s):

Mammalian Cells ◽

Ribosome Biogenesis ◽

Rrna Synthesis ◽

Dependent Manner ◽

Rrna Processing ◽

Rdna Transcription ◽

Pol I ◽

Active Transcription ◽

Key Steps ◽

Processing Factors

Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription.

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Utp14 interaction with the Small Subunit Processome

10.1101/280859 ◽

2018 ◽

Cited By ~ 1

Author(s):

Joshua J. Black ◽

Zhaohui Wang ◽

Lisa M. Goering ◽

Arlen W. Johnson

Keyword(s):

Large Scale ◽

Ribosomal Subunit ◽

Small Subunit ◽

Primary Component ◽

Uv Crosslinking ◽

The Status ◽

Rna Exosome ◽

Ssu Processome ◽

Open Question ◽

Two Hybrid

AbstractThe SSU Processome (sometimes referred to as 90S) is an early stabile intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU Processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU Processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU Processome. Thus, release of U3 is prerequisite for the transition to pre-40S. Our lab proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU Processome protein Utp14. How Utp14 times the activation of Dhr1 is an open question. Despite being highly conserved, Utp14 contains no recognizable domains, and how Utp14 interacts with the SSU Processome is not well characterized. Here, we used UV crosslinking and analysis of cDNA and yeast two-hybrid interaction to characterize how Utp14 interacts with the pre-ribosome. Moreover, proteomic analysis of SSU particles lacking Utp14 revealed that Utp14 is needed for efficient recruitment of the RNA exosome. Our analysis positions Utp14 to be uniquely poised to communicate the status of assembly of the SSU Processome to Dhr1 and possibly the exosome as well.

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DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

Molecular and Cellular Biology ◽

10.1128/mcb.01348-14 ◽

2014 ◽

Vol 35 (5) ◽

pp. 816-830 ◽

Cited By ~ 6

Author(s):

Sahar Soltanieh ◽

Yvonne N. Osheim ◽

Krasimir Spasov ◽

Christian Trahan ◽

Ann L. Beyer ◽

...

Keyword(s):

Rna Helicase ◽

Sucrose Density Gradient ◽

Small Subunit ◽

Specific Protein ◽

Rrna Synthesis ◽

Catalytic Core ◽

Dead Box ◽

Cell Extracts ◽

U3 Snorna ◽

Ssu Processome

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

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A Novel Small-Subunit Processome Assembly Intermediate That Contains the U3 snoRNP, Nucleolin, RRP5, and DBP4

Molecular and Cellular Biology ◽

10.1128/mcb.00029-09 ◽

2009 ◽

Vol 29 (11) ◽

pp. 3007-3017 ◽

Cited By ~ 41

Author(s):

Amy Jane Turner ◽

Andrew Alexander Knox ◽

José-Luis Prieto ◽

Brian McStay ◽

Nicholas James Watkins

Keyword(s):

Ribosome Biogenesis ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dynamic Structure ◽

Small Subunit ◽

Rrna Processing ◽

Key Factors ◽

Assembly Intermediate ◽

Ssu Processome

ABSTRACT Eukaryotic 18S rRNA processing is mediated by the small subunit (SSU) processome, a machine comprised of the U3 small nucleolar RNP (U3 snoRNP), tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes. We report that the human SSU processome is a dynamic structure with the recruitment and release of subcomplexes occurring during the early stages of ribosome biogenesis. A novel 50S U3 snoRNP accumulated when either pre-rRNA transcription was blocked or the tUTP proteins were depleted. This complex did not contain the tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes but was associated with the RNA-binding proteins nucleolin and RRP5 and the RNA helicase DBP4. Our data suggest that the 50S U3 snoRNP is an SSU assembly intermediate that is likely recruited to the pre-rRNA through the RNA-binding proteins nucleolin and RRP5. We predict that nucleolin is only transiently associated with the SSU processome and likely leaves the complex not long after 50S U3 snoRNP recruitment. The nucleolin-binding site potentially overlaps that of several other key factors, and we propose that this protein must leave the SSU processome for pre-rRNA processing to occur.

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