Susan J. Baserga

A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3–pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1–17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5′ ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

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.

The Small-Subunit Processome Is a Ribosome Assembly Intermediate

ABSTRACT The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5′ end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

Imp3p and Imp4p, two specific components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing.

ABSTRACT The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.

An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs.

The mammalian U3 snRNP is one member of a recently described family of nucleolar snRNPs which also includes U8, U13, U14, X and Y. All of these snRNPs are immunoprecipitable by anti‐fibrillarin autoantibodies, suggesting the existence of a common binding site for the 34 kDa fibrillarin (Fb) protein. Two short nucleotide sequences, called Boxes C and D, present in each of these RNAs are the most likely sites for fibrillarin binding. We have developed a HeLa in vitro assembly system for binding of fibrillarin to human U3 snRNA. Reconstitution of the input RNA is specific in our assay since four of the other nucleolar small RNAs (U8, U13, X and Y) which have Boxes C and D become immunoprecipitable by anti‐fibrillarin whereas two RNAs which lack these sequences (5S and 5.8S) do not. Deletion analyses of the U3 snRNA demonstrate that the presence of Box C but not Box D is required for fibrillarin binding. Moreover, seven single or double site‐specific mutations in the U3 Box C abolish binding. The role of the Box C‐fibrillarin interaction in the biogenesis of the Fb snRNPs is discussed.

Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast.

We have isolated and characterized Mpp10p, a novel protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) from the yeast Saccharomyces cerevisiae. The MPP10 protein was first identified in human cells by its reactivity with an antibody that recognizes specific sites of mitotic phosphorylation. To study the functional role of MPP10 in pre-rRNA processing, we identified the yeast protein by performing a GenBank search. The yeast Mpp10p homolog is 30% identical to the human protein over its length. Antibodies to the purified yeast protein recognize a 110-kDa polypeptide in yeast extracts and immunoprecipitate the U3 snoRNA, indicating that Mpp10p is a specific protein component of the U3 snoRNP in yeast. As a first step in the genetic analysis of Mpp10p function, diploid S. cerevisiae cells were transformed with a null allele. Sporulation and tetrad analysis indicate that MPP10 is an essential gene. A strain was constructed where Mpp10p is expressed from a galactose-inducible, glucose- repressible promoter. After depletion of Mpp10p by growth in glucose, cell growth is arrested and levels of 18S and its 20S precursor are reduced or absent while the 23S and 35S precursors accumulate. This pattern of accumulation of rRNA precursors suggests that Mpp10p is required for cleavage at sites A0, A1, and A2. Pulse-chase analysis of newly synthesized pre-rRNAs in Mpp10p-depleted yeast confirms that little mature 18S rRNA formed. These results reveal a novel protein essential for ribosome biogenesis and further elucidate the composition of the U3 snoRNP.

The small subunit processome in ribosome biogenesis—progress and prospects.

The small subunit (SSU) processome is a 2.2‐MDa ribonucleoprotein complex involved in the processing, assembly, and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloging the components is the first step toward understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into subcomplexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of subcomplex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole. WIREs RNA 2011 2 1–21 DOI: 10.1002/wrna.57

The putative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA processing through a direct interaction with Rps14

ABSTRACT One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3′ end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.

Genetics. Mysterious ribosomopathies.

Ribosomes are essential in all cell types, yet mutations to ribosomal proteins or assembly factors cause tissue-specific disease. Ribosomes are absolutely essential for life, generating all cellular proteins required for growth. The prevailing thought for many years was that mutations in ribosomal proteins or ribosome assembly factors would be lethal to developing embryos. Complete loss of any single ribosomal protein often leads to embryonic lethality in mice (1). Yet, mutations in ribosomal proteins or ribosome assembly factors result in a puzzling phenomenon—a specific mutation can affect a specific cell type and cause a tissue-specific human disease. What accounts for this tissue proclivity has been a mystery. Why do defects in a macromolecule as ubiquitous and essential as the ribosome cause diseases—ribosomopathies—only in select tissues?

Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins.

We have identified an apparent human homolog of the yeast Nop5/Nop58 protein. hNop5/Nop58 codes for a protein of predicted molecular weight 59.6 kDa and is 46.8% identical to Saccharomyces cerevisiae Nop5/Nop58. Immunofluorescent staining with antibodies against hNop5/Nop58 indicate that it is localized primarily to the nucleolus, and coimmunoprecipitation from nuclear extracts demonstrates that hNop5/Nop58 interacts with the box C/D family of snoRNAs. Thus, hNop5/Nop58 is a common component of the box C/D snoRNPs, and joins fibrillarin as the second such component identified and characterized in metazoans.