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

1997 ◽  
Vol 17 (10) ◽  
pp. 5803-5812 ◽  
Author(s):  
D A Dunbar ◽  
S Wormsley ◽  
T M Agentis ◽  
S J Baserga
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Essential Gene ◽  
Specific Protein ◽  
Protein Component ◽  
Rrna Processing ◽  
Yeast Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Phosphorylation ◽  
Novel Protein

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.

scholarly journals Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA

2020 ◽  
Vol 48 (7) ◽  
pp. 3848-3868 ◽  
Author(s):  
Guillaume Clerget ◽  
Valérie Bourguignon-Igel ◽  
Nathalie Marmier-Gourrier ◽  
Nicolas Rolland ◽  
Ludivine Wacheul ◽  
...  
Keyword(s):  
Protein Interaction Network ◽  
18S Rrna ◽  
Direct Interaction ◽  
Interaction Network ◽  
Specific Protein ◽  
Rrna Processing ◽  
Protein Protein Interaction ◽  
U3 Snorna ◽  
Ssu Processome ◽  
Protein Protein Interaction Network

Abstract U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5′-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

scholarly journals Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

1999 ◽  
Vol 19 (8) ◽  
pp. 5441-5452 ◽  
Author(s):  
Sarah J. Lee ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
18S Rrna ◽  
Rna Binding ◽  
Specific Protein ◽  
Rrna Processing ◽  
Binding Domains ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Protein Components

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.

scholarly journals Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (6) ◽  
pp. 2083-2095 ◽  
Author(s):  
Emmanuel Vanrobays ◽  
Jean-Paul Gelugne ◽  
Pierre-Emmanuel Gleizes ◽  
Michele Caizergues-Ferrer
Keyword(s):  
18S Rrna ◽  
Nuclear Protein ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Rrna Maturation ◽  
Cytoplasmic Maturation

ABSTRACT Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

scholarly journals Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

1994 ◽  
Vol 14 (9) ◽  
pp. 6361-6371 ◽  
Author(s):  
I Samejima ◽  
M Yanagida
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Gene Product ◽  
Budding Yeast ◽  
Essential Gene ◽  
Restrictive Temperature ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Novel Protein

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

Rrp9p, a novel protein component of the yeast U3 snoRNP, is required for the synthesis of 18S rRNA

2001 ◽  
Vol 1 (S1) ◽  
Author(s):  
H Vos ◽  
J Venema ◽  
AW Faber ◽  
P Bohlander ◽  
WJ van Venrooij ◽  
...  
Keyword(s):  
18S Rrna ◽  
Protein Component ◽  
Novel Protein

scholarly journals The Putative NTPase Fap7 Mediates Cytoplasmic 20S Pre-rRNA Processing through a Direct Interaction with Rps14

2005 ◽  
Vol 25 (23) ◽  
pp. 10352-10364 ◽  
Author(s):  
Sander Granneman ◽  
Madhusudan R. Nandineni ◽  
Susan J. Baserga
Keyword(s):  
High Throughput ◽  
Structural Component ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Direct Interaction ◽  
Nucleoside Triphosphate ◽  
Rrna Processing ◽  
Conserved Residues ◽  
Rrna Maturation

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.

scholarly journals Erroneous ribosomal RNAs promote the generation of antisense ribosomal siRNA

2018 ◽  
Vol 115 (40) ◽  
pp. 10082-10087 ◽  
Author(s):  
Chengming Zhu ◽  
Qi Yan ◽  
Chenchun Weng ◽  
Xinhao Hou ◽  
Hui Mao ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Rnai Pathway ◽  
Rrna Processing ◽  
Reverse Genetic ◽  
Genetic Screens ◽  
Multistep Process ◽  
Quality Control Mechanism ◽  
26S Rrna ◽  
Nuclear Rnai

Ribosome biogenesis is a multistep process, during which mistakes can occur at any step of pre-rRNA processing, modification, and ribosome assembly. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that down-regulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNAs, we conducted both forward and reverse genetic screens to search for more suppressor of siRNA (susi) mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of the 26S rRNA. C27F2.4(ceBUD23) is an m7G-methyltransferase of the 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A-methyltransferase of the 18S rRNA. Mutation of these genes led to a deficiency in modification of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasmic-to-nuclear and cytoplasmic-to-nucleolar translocations of the Argonaute protein NRDE-3. The rRNA processing deficiency also resulted in accumulation of risiRNAs. We also isolated SUSI-3(RIOK-1), which is similar to human RIOK1, that cleaves the 20S rRNA to 18S. We further utilized RNAi and CRISPR-Cas9 technologies to perform candidate-based reverse genetic screens and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently, turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain homeostasis of rRNAs.

scholarly journals Impaired ribosome biogenesis in Diamond-Blackfan anemia

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1275-1283 ◽  
Author(s):  
Valérie Choesmel ◽  
Daniel Bacqueville ◽  
Jacques Rouquette ◽  
Jacqueline Noaillac-Depeyre ◽  
Sébastien Fribourg ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Diamond Blackfan Anemia ◽  
Nucleolar Organization ◽  
Ribosomal Protein S19

Abstract The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.

scholarly journals Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

2011 ◽  
Vol 43 (14) ◽  
pp. 855-872 ◽  
Author(s):  
Ajit N. Shah ◽  
Daniela Cadinu ◽  
R. Michael Henke ◽  
Xiantong Xin ◽  
Ranita Ghosh Dastidar ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Specific Protein ◽  
Hypoxia Tolerance ◽  
Deletion Mutants ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genome Wide ◽  
A Genome ◽  
In The Wild ◽  
Induced Changes

Hypoxia is a widely occurring condition experienced by diverse organisms under numerous physiological and disease conditions. To probe the molecular mechanisms underlying hypoxia responses and tolerance, we performed a genome-wide screen to identify mutants with enhanced hypoxia tolerance in the model eukaryote, the yeast Saccharomyces cerevisiae . Yeast provides an excellent model for genomic and proteomic studies of hypoxia. We identified five genes whose deletion significantly enhanced hypoxia tolerance. They are RAI1, NSR1, BUD21, RPL20A, and RSM22, all of which encode functions involved in ribosome biogenesis. Further analysis of the deletion mutants showed that they minimized hypoxia-induced changes in polyribosome profiles and protein synthesis. Strikingly, proteomic analysis by using the iTRAQ profiling technology showed that a substantially fewer number of proteins were changed in response to hypoxia in the deletion mutants, compared with the parent strain. Computational analysis of the iTRAQ data indicated that the activities of a group of regulators were regulated by hypoxia in the wild-type parent cells, but such regulation appeared to be diminished in the deletion strains. These results show that the deletion of one of the genes involved in ribosome biogenesis leads to the reversal of hypoxia-induced changes in gene expression and related regulators. They suggest that modifying ribosomal function is an effective mechanism to minimize hypoxia-induced specific protein changes and to confer hypoxia tolerance. These results may have broad implications in understanding hypoxia responses and tolerance in diverse eukaryotes ranging from yeast to humans.

scholarly journals Role of Pre-rRNA Base Pairing and 80S Complex Formation in Subnucleolar Localization of the U3 snoRNP

2004 ◽  
Vol 24 (19) ◽  
pp. 8600-8610 ◽  
Author(s):  
Sander Granneman ◽  
Judith Vogelzangs ◽  
Reinhard Lührmann ◽  
Walther J. van Venrooij ◽  
Ger J. M. Pruijn ◽  
...  
Keyword(s):  
Complex Formation ◽  
Ribosome Biogenesis ◽  
Specific Protein ◽  
Direct Result ◽  
Rrna Processing ◽  
Base Pairing ◽  
Active Involvement ◽  
U3 Snorna ◽  
Fibrillar Component

ABSTRACT In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.

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