The Small-Subunit Processome Is a Ribosome Assembly Intermediate

Bernstein, Kara A.; Gallagher, Jennifer E. G.; Mitchell, Brianna M.; Granneman, Sander; Baserga, Susan J.

doi:10.1128/ec.3.6.1619-1626.2004

Cited by 159 publications

(160 citation statements)

References 41 publications

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“…Many of the SSU processome protein components contain protein-protein interaction domains such as WD repeats and coiled-coil domains, although there are a few identifiable known RNA-binding domains. Some SSU r-proteins can also be found associated with it (Bernstein et al 2004). However, assembly factors specific for LSU biogenesis are not found associated with the SSU processome in yeast.…”

Section: Nucleolar Pre-rrna Cleavage Steps In Pre-40s Biogenesismentioning

confidence: 97%

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

2013

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Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (.5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. TABLE OF CONTENTS Abstract 643Introduction 644

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Section: Nucleolar Pre-rrna Cleavage Steps In Pre-40s Biogenesismentioning

confidence: 97%

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

2013

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“…However, the list of proteins involved in early steps of SSU biogenesis is not complete, as new components of early pre-rRNA processing complexes continue to be identified (4,(14)(15)(16)(17). Here we add two previously undescribed proteins to that list, Utp23 and Utp24, based on the following lines of evidence: First, conditional depletion of either Utp23 or Utp24 specifically inhibits the A 0 -A 2 cleavages essential for SSU biogenesis ( Fig.…”

Section: Discussionmentioning

confidence: 99%

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Bleichert

Granneman

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A 1 and͞or A2.ribosome biogenesis ͉ small subunit processome ͉ endonuclease ͉ yeast ͉ RNA processing R ibosomes translate mRNA into proteins and, thereby, govern cell growth and survival. In Saccharomyces cerevisiae, ribosome biogenesis starts with the transcription of the rDNA into a polycistronic rRNA precursor by RNA polymerase I in the nucleolus. The primary transcript, the 35S pre-rRNA, encodes the small subunit (SSU) rRNA, 18S, the large subunit rRNAs, 25S and 5.8S, and also external and internal transcribed spacer regions (5ЈETS, ITS1, ITS2, and 3ЈETS; Fig. 1). This precursor is extensively endoand exonucleolytically cleaved to mature the rRNAs. Maturation begins with endonucleolytic cleavages at sites A 0 , A 1 , and A 2 , the latter separating the 20S pre-rRNA, the direct precursor to the mature 18S rRNA, and the 27SA 2 pre-rRNA, the precursor to the large subunit rRNAs. The 20S pre-rRNA is exported into the cytoplasm as part of the 43S preribosome, where the 18S rRNA is matured by cleavage at site D.The A 0 , A 1 , and A 2 pre-rRNA cleavages require a large ribonucleoprotein complex, the SSU processome͞90S preribosome, which contains at least 40 different proteins and numerous small nucleolar RNAs (snoRNAs) (1-4). This complex likely corresponds to the terminal knobs seen in Miller chromatin spreads (1, 5). Depletion of essential SSU processome components prevents cleavages at sites A 0 -A 2 but not A 3 . Although this results in accumulation of the 23S pre-rRNA and inhibition of 18S synthesis, maturation of the 25S and 5.8S rRNAs can occur normally (Fig. 1).Despite the identification of numerous proteins involved in early pre-18S rRNA processing by large-scale purifications, the endonuclease(s) and possible candidates that carry out these cleavages have remained elusive. Recently, an interesting group of proteins possessing a PIN domain (PilT N terminus, PINc domain in SMART database) has been impl...

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“…Yeasts harboring plasmids with wild-type or mutated RNA helicases were shifted into SG-URA medium for 6 h. Two milliliters of culture at an OD 600 value of 0.4 was pelleted, and protein was extracted as previously described (29). Protein was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted with peroxidase-antiperoxidase antibodies (PAP; Sigma) to detect the protein A portion of the TAP tag and anti-Mpp10 antibodies as previously described (3,4).…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Bernstein

Granneman²,

Lee³

et al. 2006

Molecular and Cellular Biology

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DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.Virtually every cellular process involving RNA requires the function of an ATP-dependent DEXD/H box RNA helicase. These enzymes are known to be involved in mRNA splicing, transcription, RNA editing, and ribosome biogenesis. RNA helicases are conserved from bacteria to humans and require eight conserved motifs for ATP binding and hydrolysis, substrate binding, and conformational changes related to their functions ( Fig. 1A) (11,15,16,42,45,48). In particular, motif I, motif II (DEXD/H box), and motif VI are required for ATPase activity; motif III (SAT) couples ATP hydrolysis with helicase function and motif VI is also required for RNA interactions. Recently, it has been shown that RNA helicases, in addition to their ability to bind and unwind RNA, can also modify ribonucleoprotein complexes (RNPs) (19,25).Pre-rRNA processing begins with transcription of the 35S pre-rRNA in the nucleolus. Through a stepwise series of cleavage events, this transcript is processed into the mature 18S, 5.8S, and 25S rRNAs (Fig. 1B). The 5S rRNA is transcribed and processed separately. The 5S, 5.8S, and 25S rRNAs become incorporated into the large ribosomal subunit (LSU), while the 18S becomes incorporated into the small ribosomal subunit (SSU). Processing in internal transcribed spacer 1 separates the small and large ribosomal precursors and forms the 20S and 27SA 2 pre-rRNAs (Fig. 1B). The 20S pre-rRNA is subsequently processed at site D to form the mature 18S rRNA. Processing of the 5.8S and 25S rRNAs occurs through two distinct pathways. In the major pathway, the 27SA 2 prerRNA is subsequently cleaved into the...

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The Small-Subunit Processome Is a Ribosome Assembly Intermediate

Cited by 159 publications

References 41 publications

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

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