The Complexity of Human Ribosome Biogenesis Revealed by Systematic Nucleolar Screening of Pre-rRNA 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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Potential Roles for Ubiquitin and the Proteasome during Ribosome Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.02227-05 ◽

2006 ◽

Vol 26 (13) ◽

pp. 5131-5145 ◽

Cited By ~ 62

Author(s):

Diana A. Stavreva ◽

Miyuki Kawasaki ◽

Miroslav Dundr ◽

Karel Koberna ◽

Waltraud G. Müller ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Ribosome Biogenesis ◽

Dynamic Properties ◽

Proteasome Inhibition ◽

Ubiquitin Proteasome System ◽

Rrna Processing ◽

Proteasome Degradation ◽

Ubiquitin Proteasome ◽

Processing Factors

ABSTRACT We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

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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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Ribonucleoproteins in Archaeal Pre-rRNA Processing and Modification

Archaea ◽

10.1155/2013/614735 ◽

2013 ◽

Vol 2013 ◽

pp. 1-14 ◽

Cited By ~ 13

Author(s):

W. S. Vincent Yip ◽

Nicholas G. Vincent ◽

Susan J. Baserga

Keyword(s):

Ribosome Biogenesis ◽

Gene Organization ◽

Rrna Gene ◽

Rrna Processing ◽

Ribosomal Rnas ◽

Intensive Research ◽

Complex Process ◽

Nucleotide Modification

Given that ribosomes are one of the most important cellular macromolecular machines, it is not surprising that there is intensive research in ribosome biogenesis. Ribosome biogenesis is a complex process. The maturation of ribosomal RNAs (rRNAs) requires not only the precise cleaving and folding of the pre-rRNA but also extensive nucleotide modifications. At the heart of the processing and modifications of pre-rRNAs in Archaea and Eukarya are ribonucleoprotein (RNP) machines. They are called small RNPs (sRNPs), in Archaea, and small nucleolar RNPs (snoRNPs), in Eukarya. Studies on ribosome biogenesis originally focused on eukaryotic systems. However, recent studies on archaeal sRNPs have provided important insights into the functions of these RNPs. This paper will introduce archaeal rRNA gene organization and pre-rRNA processing, with a particular focus on the discovery of the archaeal sRNP components, their functions in nucleotide modification, and their structures.

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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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Post-transcriptional regulation of ribosome formation in the nucleus of regenerating rat liver

Biochemical Journal ◽

10.1042/bj2100183 ◽

1983 ◽

Vol 210 (1) ◽

pp. 183-192 ◽

Cited By ~ 7

Author(s):

K P Dudov ◽

M D Dabeva

Keyword(s):

Rat Liver ◽

Ribosome Biogenesis ◽

Rrna Processing ◽

Regenerating Liver ◽

Regenerating Rat Liver ◽

Rrna Species ◽

Rrna Maturation ◽

The Individual ◽

Post Transcriptional Regulation

Kinetic experiments on RNA labelling in vivo with [14C]orotate were performed with normal and 12h-regenerating rat liver. The specific radioactivities of nucleolar, nucleoplasmic and cytoplasmic rRNA species were analysed by computer according to the models of rRNA processing and nucleo-cytoplasmic migration given previously [Dudov, Dabeva, Hadjiolov & Todorov, Biochem. J. (1978) 171, 375-383]. The rates of formation and the half-lives of the individual pre-rRNA and rRNA species were determined in both normal and regenerating liver. The results show clearly that the formation of ribosomes in regenerating rat liver is post-transcriptionally activated: (a) the half-lives of all the nucleolar pre-rRNA and rRNA species are decreased by 30% on average; (b) the pre-rRNA processing is directed through the shortest maturation pathway: 45 S leads to 32 S + 18 S leads to 28 S; (c) the nucleo-cytoplasmic transfer of ribosomes is accelerated. As a consequence, the time for formation and appearance of ribosomes in the cytoplasm is shortened 1.5-fold for the large and 2-fold for the small subparticle. A new scheme for endonuclease cleavage of 45 S pre-rRNA is proposed, which explains the alterations in pre-rRNA processing in regenerating liver. Its validity for pre-rRNA processing in other eukaryotes is discussed. It is concluded that: (i) the control sites in the intranucleolar formation of 28 S and 18 S rRNA are the immediate precursor of 28 S rRNA, 32 S pre-rRNA, and the primary pre-rRNA, 45 S pre-rRNA, respectively; (ii) the limiting step in the post-transcriptional stages of ribosome biogenesis is the pre-rRNA maturation.

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Translational control during cellular senescence

Molecular and Cellular Biology ◽

10.1128/mcb.00512-20 ◽

2020 ◽

Author(s):

Matthew J. Payea ◽

Carlos Anerillas ◽

Ravi Tharakan ◽

Myriam Gorospe

Keyword(s):

Translational Control ◽

Ribosome Biogenesis ◽

Rrna Processing ◽

Proliferating Cells ◽

Cycle Arrest ◽

Nucleolar Stress ◽

Specific Proteins ◽

Inflammatory Proteins ◽

Secretory Phenotype

Senescence is a state of long-term cell-cycle arrest that arises in cells that have incurred sub-lethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence associated secretory phenotype (SASP). On the other hand, these pro-growth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.

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Controlling the material properties and rRNA processing function of the nucleolus using light

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903870116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17330-17335 ◽

Cited By ~ 17

Author(s):

Lian Zhu ◽

Tiffany M. Richardson ◽

Ludivine Wacheul ◽

Ming-Tzo Wei ◽

Marina Feric ◽

...

Keyword(s):

Phase Behavior ◽

Ribosomal Rna ◽

Material Properties ◽

Viscoelastic Properties ◽

Ribosome Biogenesis ◽

Threshold Concentration ◽

Rrna Processing ◽

Small Proteins ◽

Nucleolar Material ◽

Processing Steps

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.

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Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins

The Journal of Cell Biology ◽

10.1083/jcb.200612048 ◽

2007 ◽

Vol 177 (4) ◽

pp. 573-578 ◽

Cited By ~ 38

Author(s):

Tim Krüger ◽

Hanswalter Zentgraf ◽

Ulrich Scheer

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Live Cell Imaging ◽

Ribosome Biogenesis ◽

Cell Imaging ◽

Rrna Processing ◽

Dense Fibrillar Component ◽

Assembly Pathway ◽

Fibrillar Component ◽

Processing Steps

Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC–GC interface.

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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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