Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian 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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Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

RNA ◽

10.1261/rna.079025.121 ◽

2021 ◽

pp. rna.079025.121

Author(s):

Joshua J Black ◽

Arlen W Johnson

Keyword(s):

Binding Site ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Assembly Line ◽

Small Subunit ◽

Nucleotide Hydrolysis ◽

Ribonucleoprotein Complexes ◽

Intermediate States ◽

Ssu Processome

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

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Attenuation of Bunyavirus Replication by Rearrangement of Viral Coding and Noncoding Sequences

Journal of Virology ◽

10.1128/jvi.79.11.6940-6946.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6940-6946 ◽

Cited By ~ 32

Author(s):

Anice C. Lowen ◽

Amanda Boyd ◽

John K. Fazakerley ◽

Richard M. Elliott

Keyword(s):

Mammalian Cells ◽

Reverse Genetics ◽

Viral Rna ◽

Wild Type Virus ◽

Coding Region ◽

Reading Frame ◽

Ribonucleoprotein Complexes ◽

The Family ◽

Prototype Virus ◽

Negative Sense

ABSTRACT Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

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Targeted Comparative RNA Interference Analysis Reveals Differential Requirement of Genes Essential for Cell Proliferation

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0340 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4837-4845 ◽

Cited By ~ 9

Author(s):

Yuichi J. Machida ◽

Yuefeng Chen ◽

Yuka Machida ◽

Ankit Malhotra ◽

Sukumar Sarkar ◽

...

Keyword(s):

Rna Interference ◽

Cell Line ◽

Cell Lines ◽

Mammalian Cells ◽

Ribosome Biogenesis ◽

Cancer Cell Line ◽

Prostate Cancer Cell Line ◽

Breast Epithelial Cell ◽

Epithelial Cell Line ◽

Dependent Manner

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53−) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type–specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

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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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Metabolic turnover and dynamics of RNA modifications by 13C labelling

10.21203/rs.3.rs-109009/v1 ◽

2021 ◽

Author(s):

Paulo A Gameiro ◽

Iosifina Foskolou ◽

Vesela Encheva ◽

Mariana Silva dos Santos ◽

James MacRae ◽

...

Keyword(s):

T Cell Activation ◽

Mammalian Cells ◽

Quantitative Methods ◽

Cell Activation ◽

Metabolic Stress ◽

Rna Modifications ◽

Biological Regulation ◽

Rna Methylation ◽

Ribonucleoprotein Complexes ◽

Kinetics Of

Abstract RNA methylation is essential for appropriate assembly of ribonucleoprotein complexes. Dynamics of RNA methylation is thus important, but we lack quantitative methods to comprehensively assess it. We developed 13C-dynamods, an isotopic labelling approach using 13C-methyl-methionine, to quantify the turnover of base modifications in newly synthesized RNA, which is effective in distinguishing modifications in mRNAs from those in ncRNAs. This approach detected the presence of N6,N6-dimethyladenosine (m62A) in mRNA and tRNA in mammalian cells, and uncovered distinct kinetics of N-6-methyladenosine (m6A) and N-7-methylguanosine (m7G) in mRNA. Moreover, by assessing RNA metabolism during T-cell activation, we showed how methylation dynamics is coordinated with ribonucleotide biosynthesis. Finally, by quantification of methylation turnover and ribonucleoside abundance, we uncover the post-transcriptional lability of m6A in response to metabolic stress. Thus, 13C-dynamods enables studies of the origin, maintenance and biological regulation of RNA modifications under steady-state and non-stationary conditions.

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The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells

Biomolecules ◽

10.3390/biom9090422 ◽

2019 ◽

Vol 9 (9) ◽

pp. 422 ◽

Cited By ~ 1

Author(s):

Leetsi ◽

Õunap ◽

Abroi ◽

Kurg

Keyword(s):

Mammalian Cells ◽

Ribosome Biogenesis ◽

Substrate Binding ◽

Mrna Level ◽

Binding Motif ◽

Protein Levels ◽

Histone Lysine Methyltransferase ◽

Lysine Methyltransferase ◽

Alternatively Spliced ◽

The Common

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

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The young and the restless: Isolating the dynamic mammalian preribosomes

Journal of Biological Chemistry ◽

10.1074/jbc.h119.009702 ◽

2019 ◽

Vol 294 (28) ◽

pp. 10758-10759

Author(s):

Estela Jacinto

Keyword(s):

Structural Analysis ◽

Genome Editing ◽

Ribosome Biogenesis ◽

Affinity Purification ◽

Ribonucleoprotein Complexes ◽

Purification Scheme

Despite the advances in understanding the assembly of yeast preribosomes using affinity purification and structural analysis, studies on mammalian ribosome biogenesis have lagged behind. Using an unbiased method to purify native mammalian preribosomal complexes from the nucleus, Abetov et al. now uncover two types of premature ribonucleoprotein complexes that are nutrient- and mTOR-dependent. This purification scheme, combined with genome-editing techniques, could be exploited to untangle the complexities underlying human ribosome biogenesis and ribosomopathies.

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Unbalanced X Chromosome Inactivation Independent of Telomere Shortening in Mice Heterozygous for a Mutant Dyskerin Allele.

Blood ◽

10.1182/blood.v108.11.184.184 ◽

2006 ◽

Vol 108 (11) ◽

pp. 184-184

Author(s):

Baiwei Gu ◽

Jun He ◽

Monica Bessler ◽

Philip J. Mason

Keyword(s):

Bone Marrow ◽

Ribosome Biogenesis ◽

Es Cells ◽

X Inactivation ◽

Telomere Maintenance ◽

Male Mice ◽

Wild Type ◽

Telomere Shortening ◽

Ribonucleoprotein Complexes ◽

The Impact

Abstract X-linked Dyskeratosis Congenita (DC) is a rare recessive disorder caused by mutations in the DKC1 gene that encodes dyskerin. Dyskerin is part of ribonucleoprotein complexes that participate in two different pathways: ribosome biogenesis and telomere maintenance. It is the subject of intense debate whether disease manifestations in DC are due to dysfunctional telomere maintenances or are caused by a defect in ribosome biogenesis. Pathogenic mutations in dyskerin cause telomere shortening and patients with X-linked DC have critically short telomeres, However, whether there is an additional defect in ribosome biogenesis is difficult to investigate. To dissect the impact of a pathogenic dyskerin mutation on telomeres from the possible additional impact on ribosome biogenesis in an in vivo model, we generated mice expressing a mutant dyskerin protein. Because laboratory mice have very long telomeres a short telomere phenotype requires several generations of inbreeding, whereas a phenotype seen in the first generation is likely to be caused by the defect in ribosome biogenesis. To delete the last 21 amino acids of dyskerin (Del15) we used homologous recombination followed by conditional gene deletion in murine embryonic stem (ES) cells and in mice. Six independent ES cell clones with the deleted Dkc1 gene were obtained. In vitro analysis of the ES cells showed that the Del15 mutation led to dramatically decreased expression of a truncated dyskerin protein with decreased accumulation of the telomerase RNA. In addition, both reduction in telomerase activity and significant telomere shortening after 65 passages were observed. These findings indicate that the Del15 mutation impairs the telomerase maintenance pathway. After testing the accumulation of a series of mouse H/ACA snoRNA in Del15 ES cells, we found a decrease of the mU68 and mE1 snoRNAs suggesting the mutation may also confer effects which are outside the telomerase pathway. We therefore went on to produce a line of mice expressing the truncated Dkc1 protein and were able to obtain male mice hemizygous for the mutant Dkc1 gene as well as female heterozgotes. The male mice express the truncated dyskerin protein and show no gross abnormality up to 6 months of age. Interestingly, heterozygous female mice were healthy as well but the truncated dyskerin protein was dramatically decreased in expression compared to the wild type dyskerin in spleen, thymus, and bone marrow, but not in liver and brain. This result must derive from preferential proliferation of cells expressing wild type dyskerin after random X-inactivation in early embryogenesis. Our analysis indicates that the mutant dyskerin impairs the proliferation in hematopoietic tissues while it does not affect cells which are not rapidly proliferating such as those in liver and brain. Because of the early appearance of the skewed X-inactivation phenotype we conclude that skewing in these mice is caused by a telomere independent mechanism. Interestingly, the lack of overt DC-like abnormalities in the male hemizygous mice indicates that this proliferative disadvantage is insufficient to cause bone marrow failure but in combination with impaired telomere maintenance may accelerate the onset and severity of disease and thus explain the earlier and more severe manifestation in X-linked DC compared to autosomal dominant DC which only affects the telomerase pathway.

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Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0249 ◽

2007 ◽

Vol 18 (2) ◽

pp. 394-403 ◽

Cited By ~ 29

Author(s):

K. Kopp ◽

J. Z. Gasiorowski ◽

D. Chen ◽

R. Gilmore ◽

J. T. Norton ◽

...

Keyword(s):

Mammalian Cells ◽

Ribosome Biogenesis ◽

Rrna Synthesis ◽

Dependent Manner ◽

Rrna Processing ◽

Rdna Transcription ◽

Pol I ◽

Active Transcription ◽

Key Steps ◽

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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