Las1L Is a Nucleolar Protein Required for Cell Proliferation and Ribosome Biogenesis

ABSTRACT Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.

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hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA.

10.1101/2021.11.12.468419 ◽

2021 ◽

Author(s):

Han Liao ◽

Anushri Gaur ◽

Hunter McConie ◽

Amirtha Shekar ◽

Karen Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Complex Formation ◽

Ribosome Biogenesis ◽

Bisulfite Sequencing ◽

28S Rrna ◽

Rrna Processing ◽

Catalytic Complex ◽

Base Modification ◽

Rrna Depletion ◽

First Time

5-Methylcytosine (m5C) is a base modification broadly found on a variety of RNAs in the human transcriptome. In eukaryotes m5C is catalyzed by enzymes of the NSUN family, which is composed of seven members in humans (NSUN1-7). NOP2/NSUN1 has been mostly characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S rRNA. Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions remain poorly characterized. To define the roles of human NOP2/NSUN1, we used an miCLIP-seq approach to identify its RNA substrates. Our analysis reveals that vault RNA 1.2 and rRNA are NOP2/NSUN1-specific methylated targets and we further confirm by bisulfite sequencing that NOP2/NSUN1 is responsible for the deposition of m5C at residue 4447 on the 28S rRNA. Depletion of NOP2/NSUN1 impairs cell proliferation, rRNA processing and 60S ribosome biogenesis. Additionally, we find that NOP2/NSUN1 binds to the 5′ETS region of the pre-rRNA transcript and regulates pre-rRNA processing in part through non-catalytic complex formation with box C/D snoRNAs. Our study identifies for the first time the RNA substrates of human NOP2/NSUN1 and reveals additional functions in rRNA processing beyond catalyzing m5C base modification.

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Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5516-5528.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5516-5528 ◽

Cited By ~ 117

Author(s):

Žaklina Strezoska ◽

Dimitri G. Pestov ◽

Lester F. Lau

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Weight Fraction ◽

Rnase A ◽

Mutant Form ◽

Rrna Processing ◽

Ribonucleoprotein Complexes ◽

Nuclear Extracts ◽

Mouse Tissues ◽

Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

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Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614852113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11967-11972 ◽

Cited By ~ 7

Author(s):

Pan Zhu ◽

Yuqiu Wang ◽

Nanxun Qin ◽

Feng Wang ◽

Jia Wang ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Higher Plants ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Developmental Defects ◽

Protein Mutants ◽

5.8S Rrna ◽

Important Regulator ◽

Nucleolar Rna

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA–rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

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Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1518 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1518-1525 ◽

Cited By ~ 60

Author(s):

Nilson I. T. Zanchin ◽

David S. Goldfarb

Keyword(s):

Ribosome Biogenesis ◽

18S Rrna ◽

Fluorescent Protein ◽

Protein A ◽

Nucleolar Protein ◽

Mrna Turnover ◽

5.8S Rrna ◽

Multistep Process ◽

Green Fluorescent ◽

Two Hybrid

ABSTRACT NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.

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Bystin in human cancer cells: intracellular localization and function in ribosome biogenesis

Biochemical Journal ◽

10.1042/bj20061597 ◽

2007 ◽

Vol 404 (3) ◽

pp. 373-381 ◽

Cited By ~ 25

Author(s):

Masaya Miyoshi ◽

Tetsuya Okajima ◽

Tsukasa Matsuda ◽

Michiko N. Fukuda ◽

Daita Nadano

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Ribosomal Rna ◽

Mammalian Cells ◽

Ribosome Biogenesis ◽

Human Cancer ◽

Intracellular Localization ◽

Promote Cell Proliferation ◽

Human Cancer Cell Lines ◽

Nucleolar Stress

Although bystin has been identified as a protein potentially involved in embryo implantation (a process unique to mammals) in humans, the bystin gene is evolutionarily conserved from yeast to humans. DNA microarray data indicates that bystin is overexpressed in human cancers, suggesting that it promotes cell growth. We undertook RT (reverse transcription)–PCR and immunoblotting, and confirmed that bystin mRNA and protein respectively are expressed in human cancer cell lines, including HeLa. Subcellular fractionation identified bystin protein as nuclear and cytoplasmic, and immunofluorescence showed that nuclear bystin localizes mainly in the nucleolus. Sucrose gradient ultracentrifugation of total cytoplasmic ribosomes revealed preferential association of bystin with the 40S subunit fractions. To analyse its function, bystin expression in cells was suppressed by RNAi (RNA interference). Pulse–chase analysis of ribosomal RNA processing suggested that bystin knockdown delays processing of 18S ribosomal RNA, a component of the 40S subunit. Furthermore, this knockdown significantly inhibited cell proliferation. Our findings suggest that bystin may promote cell proliferation by facilitating ribosome biogenesis, specifically in the production of the 40S subunit. Localization of bystin to the nucleolus, the site of ribosome biogenesis, was blocked by low concentrations of actinomycin D, a reagent that causes nucleolar stress. When bystin was transiently overexpressed in HeLa cells subjected to nucleolar stress, nuclear bystin was included in particles different from the nuclear stress granules induced by heat shock. In contrast, cytoplasmic bystin was barely affected by nucleolar stress. These results suggest that, while bystin may play multiple roles in mammalian cells, a conserved function is to facilitate ribosome biogenesis required for cell growth.

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Small nucleolar RNA

Biochemistry and Cell Biology ◽

10.1139/o95-092 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 845-858 ◽

Cited By ~ 27

Author(s):

Susan A. Gerbi

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Ribosome Biogenesis ◽

Internal Transcribed Spacers ◽

28S Rrna ◽

Small Nucleolar Rnas ◽

Rrna Processing ◽

Conserved Sequence ◽

Nucleolar Rna ◽

Nucleolar Rnas

A growing list of small nucleolar RNAs (snoRNAs) has been characterized in eukaryotes. They are transcribed by RNA polymerase II or III; some snoRNAs are encoded in the introns of other genes. The nonintronic polymerase II transcribed snoRNAs receive a trimethylguanosine cap, probably in the nucleus, and move to the nucleolus. snoRNAs are complexed with proteins, sometimes including fibrillarin. Localization and maintenance in the nucleolus of some snoRNAs requires the presence of initial precursor rRNA (pre-rRNA). Many snoRNAs have conserved sequence boxes C and D and a 3′ terminal stem; the roles of these features are discussed. Functional assays done for a few snoRNAs indicate their roles in rRNA processing for cleavage of the external and internal transcribed spacers (ETS and ITS). U3 is the most abundant snoRNA and is needed for cleavage of ETS1 and ITS1; experimental results on U3 binding sites in pre-rRNA are reviewed. 18S rRNA production also needs U14, U22, and snR30 snoRNAs, whereas U8 snoRNA is needed for 5.8S and 28S rRNA production. Other snoRNAs that are complementary to 18S or 28S rRNA might act as chaperones to mediate RNA folding. Whether snoRNAs join together in a large rRNA processing complex (the "processome") is not yet clear. It has been hypothesized that such complexes could anchor the ends of loops in pre-rRNA containing 18S or 28S rRNA, thereby replacing base-paired stems found in pre-rRNA of prokaryotes.Key words: RNA processing, small nucleolar RNAs, nucleolus, ribosome biogenesis, rRNA processing complex.

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Sfp1 Plays a Key Role in Yeast Ribosome Biogenesis

Eukaryotic Cell ◽

10.1128/ec.2.5.1061-1068.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 1061-1068 ◽

Cited By ~ 72

Author(s):

Ian Fingerman ◽

Vijayalakshmi Nagaraj ◽

David Norris ◽

Andrew K. Vershon

Keyword(s):

Ribosome Biogenesis ◽

Zinc Finger Protein ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Reporter Construct ◽

Promoter Elements ◽

Translational Machinery ◽

Finger Protein ◽

Activation Of Transcription ◽

Direct Binding

ABSTRACT Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect. sfp1 cells have a reduced size and a slow growth phenotype. In this study we show that SFP1 plays a role in ribosome biogenesis. An sfp1 strain is hypersensitive to drugs that inhibit translational machinery. sfp1 strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed SFP1 induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is SFP1 dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.

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A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc

Development ◽

10.1242/dev.125.18.3571 ◽

1998 ◽

Vol 125 (18) ◽

pp. 3571-3584 ◽

Cited By ~ 4

Author(s):

S. Zaffran ◽

A. Chartier ◽

P. Gallant ◽

M. Astier ◽

N. Arquier ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Ribosome Biogenesis ◽

Protein Biosynthesis ◽

Ectopic Expression ◽

Rna Helicase ◽

Potential Target ◽

Dead Box ◽

First Instar ◽

Cell Growth And Proliferation

This article describes the characterization of a new Drosophila gene that we have called pitchoune (pit) (meaning small in Provence) because mutations in this gene produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7–10 days. All the tissues are equally affected and the perfectly shaped larvae are indistinguishable from first instar wild-type animals. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. Tagged-Pit, when transfected in S2 cells, localizes mainly to the nucleolus, pointing towards a possible role in ribosome biogenesis and, consequently, in protein biosynthesis. pit encodes a DEAD-box RNA helicase, a family of proteins involved in the control of RNA structure in many cellular processes and its closest homologue is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers (Grandori, C., Mac, J., Siebelt, F., Ayer, D. E. and Eisenman, R. N. (1996) EMBO J. 15, 4344–4357). The patterns of expression of d-myc and pit are superimposable. Ectopic expression of myc in the nervous system drives an ectopic expression of pit in this tissue indicating that in Drosophila as well, pit is a potential target of d-Myc. These results suggest that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation.

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RelA Functionally Suppresses the Growth Defect Caused by a Mutation in the G Domain of the Essential Der Protein

Journal of Bacteriology ◽

10.1128/jb.01758-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3236-3243 ◽

Cited By ~ 13

Author(s):

Jihwan Hwang ◽

Masayori Inouye

Keyword(s):

Cell Growth ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Synthetic Activity ◽

Growth Defect ◽

Multicopy Plasmid ◽

Ribosomal Subunits ◽

E Coli ◽

Binding Domains ◽

Gtp Binding

ABSTRACT A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.

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The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3702 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3702-3713 ◽

Cited By ~ 48

Author(s):

B A Peculis

Keyword(s):

Ribosome Biogenesis ◽

Critical Role ◽

Functional Site ◽

28S Rrna ◽

Rrna Processing ◽

Hybrid Molecules ◽

Evolutionarily Conserved ◽

Rrna Maturation ◽

Base Pairing Interaction

Ribosome biogenesis in eucaryotes involves many small nucleolar ribonucleoprotein particles (snoRNP), a few of which are essential for processing pre-rRNA. Previously, U8 snoRNA was shown to play a critical role in pre-rRNA processing, being essential for accumulation of mature 28S and 5.8S rRNAs. Here, evidence which identifies a functional site of interaction on the U8 RNA is presented. RNAs with mutations, insertions, or deletions within the 5'-most 15 nucleotides of U8 do not function in pre-rRNA processing. In vivo competitions in Xenopus oocytes with 2'O-methyl oligoribonucleotides have confirmed this region as a functional site of a base-pairing interaction. Cross-species hybrid molecules of U8 RNA show that this region of the U8 snoRNP is necessary for processing of pre-rRNA but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the structure or proteins comprising, or recruited by, the U8 snoRNP modulate the efficiency of cleavage. Intriguingly, these 15 nucleotides have the potential to base pair with the 5' end of 28S rRNA in a region where, in the mature ribosome, the 5' end of 28S interacts with the 3' end of 5.8S. The 28S-5.8S interaction is evolutionarily conserved and critical for pre-rRNA processing in Xenopus laevis. Taken together these data strongly suggest that the 5' end of U8 RNA has the potential to bind pre-rRNA and in so doing, may regulate or alter the pre-rRNA folding pathway. The rest of the U8 particle may then facilitate cleavage or recruitment of other factors which are essential for pre-rRNA processing.

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