Publications (16)108.04 Total impact
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Article: NOL11, implicated in the pathogenesis of North American Indian childhood cirrhosis, is required for pre-rRNA transcription and processing.
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ABSTRACT: The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in baker's yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs. Our objective was to identify non-conserved interaction partners for the human ribosome biogenesis factor, hUTP4/Cirhin, since the R565W mutation in the C-terminus of hUTP4/Cirhin was reported to cause North American Indian childhood cirrhosis (NAIC). By screening a yeast two-hybrid cDNA library derived from human liver, and through affinity purification followed by mass spectrometry, we identified an uncharacterized nucleolar protein, NOL11, as an interaction partner for hUTP4/Cirhin. Bioinformatic analysis revealed that NOL11 is conserved throughout metazoans and their immediate ancestors but is not found in any other phylogenetic groups. Co-immunoprecipitation experiments show that NOL11 is a component of the human ribosomal small subunit (SSU) processome. siRNA knockdown of NOL11 revealed that it is involved in the cleavage steps required to generate the mature 18S rRNA and is required for optimal rDNA transcription. Furthermore, abnormal nucleolar morphology results from the absence of NOL11. Finally, yeast two-hybrid analysis shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin and that the R565W mutation partially disrupts this interaction. We have therefore identified NOL11 as a novel protein required for the early stages of ribosome biogenesis in humans. Our results further implicate a role for NOL11 in the pathogenesis of NAIC.PLoS Genetics 08/2012; 8(8):e1002892. · 8.69 Impact Factor -
Article: Replication timing of pseudo-NORs.
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ABSTRACT: In mammalian cells, transcriptionally active ribosomal genes are replicated in the early S phase, and the silent ribosomal genes in the late S phase, though mechanisms of this timing remain unknown. UBF (Upstream Binding Factor), a DNA binding protein and component of the pol I transcription machinery, is considered to be responsible for the loose chromatin structure of the active rDNA. Here we question whether such structure alone can ensure early replication of DNA. We investigate this problem on the model of pseudo-NORs, the tandem arrays of heterologous DNA sequence with high affinity for UBF, introduced into human chromosomes. Such arrays are not transcribed, yet efficiently bind UBF and mimic the chromatin structure of active rDNA. In our study, a human derived stable cell line containing one pseudo-NOR on the chromosome 10 was transiently transfected with UBF-GFP and PCNA-RFP, which allowed us to observe in vivo the growth of pseudo-NORs resulted from their replication. We found that replication of pseudo-NORs is not restricted to the early S phase, but continues in the late S phase at a significant level. These results were confirmed in the experiments with incorporation of thymidin analog EdU and BrdU ChIP assay. Similar results were obtained with another cell line containing pseudo-NOR on the chromosome 7. Our data indicate that the specific loose structure of chromatin, produced by the architect protein UBF, is not sufficient for the early replication.Journal of Structural Biology 02/2011; 173(2):213-8. · 3.41 Impact Factor -
Article: A novel small-subunit processome assembly intermediate that contains the U3 snoRNP, nucleolin, RRP5, and DBP4.
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ABSTRACT: Eukaryotic 18S rRNA processing is mediated by the small subunit (SSU) processome, a machine comprised of the U3 small nucleolar RNP (U3 snoRNP), tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes. We report that the human SSU processome is a dynamic structure with the recruitment and release of subcomplexes occurring during the early stages of ribosome biogenesis. A novel 50S U3 snoRNP accumulated when either pre-rRNA transcription was blocked or the tUTP proteins were depleted. This complex did not contain the tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes but was associated with the RNA-binding proteins nucleolin and RRP5 and the RNA helicase DBP4. Our data suggest that the 50S U3 snoRNP is an SSU assembly intermediate that is likely recruited to the pre-rRNA through the RNA-binding proteins nucleolin and RRP5. We predict that nucleolin is only transiently associated with the SSU processome and likely leaves the complex not long after 50S U3 snoRNP recruitment. The nucleolin-binding site potentially overlaps that of several other key factors, and we propose that this protein must leave the SSU processome for pre-rRNA processing to occur.Molecular and cellular biology 04/2009; 29(11):3007-17. · 6.06 Impact Factor -
Article: Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.
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ABSTRACT: Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.Journal of Cell Science 02/2009; 122(Pt 4):489-98. · 6.11 Impact Factor -
Article: Pseudo-NORs: a novel model for studying nucleoli.
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ABSTRACT: Nucleolar organiser regions (NORs) are comprised of tandem arrays of ribosomal gene (rDNA) repeats that are transcribed by RNA polymerase I (Pol I), ultimately resulting in formation of a nucleolus. Upstream binding factor (UBF), a DNA binding protein and component of the Pol I transcription machinery, binds extensively across the rDNA repeat in vivo. Pseudo-NORs are tandem arrays of a heterologous DNA sequence with high affinity for UBF introduced into human chromosomes. In this review we describe how analysis of pseudo-NORs has provided important insights into nucleolar formation. Pseudo-NORs mimic endogenous NORs in a number of important respects. On metaphase chromosomes both appear as secondary constrictions comprised of undercondensed chromatin. The transcriptional silence of pseudo-NORs provides a platform for studying the transcription independent recruitment of factors required for nucleolar formation by this specialised chromatin structure. During interphase, pseudo-NORs appear as distinct and novel sub-nuclear bodies. Analysis of these bodies and comparison to their endogenous counterpart has provided insights into nucleolar formation and structure.Biochimica et Biophysica Acta 08/2008; 1783(11):2116-23. · 4.66 Impact Factor -
Article: The epigenetics of rRNA genes: from molecular to chromosome biology.
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ABSTRACT: In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.Annual Review of Cell and Developmental Biology 08/2008; 24:131-57. · 15.84 Impact Factor -
Article: Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells.
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ABSTRACT: Efficient ribosome biogenesis requires coordination of a highly complex series of events. Early events include pre-RNA transcription, processing, and modification. Analysis in yeast has demonstrated that t-UTPs, components of the U3 snoRNA-containing pre-rRNA processing complex, are required for efficient transcription of ribosomal genes (rDNA) by RNA polymerase I (pol I). Here, we characterize human t-UTPs and establish that their ability to link transcription and pre-rRNA processing is evolutionarily conserved. The pol I transcription factor UBF binds extensively across rDNA throughout the cell cycle, resulting in a specialized form of chromatin that is the hallmark of active nucleolar organizer regions (NORs). Transcriptionally silent pseudo-NORs are ectopic, chromosomally integrated, artificial arrays that mimic this specialized chromatin structure. Pseudo-NORs sequester t-UTPs and factors linking transcription with pre-rRNA modification (Nopp140 and Treacle). Recruitment is independent of transcription, the underlying DNA sequence, and location within the nucleolus. Previously, we have demonstrated that pseudo-NORs sequester every component of the pol I transcription machinery. Taken together, these results highlight the importance of the specialized chromatin structure at active NORs in coordinating early events in ribosome biogenesis and nucleolar formation.Genes & Development 09/2007; 21(16):2041-54. · 11.66 Impact Factor -
Article: Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis.
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ABSTRACT: When human adenovirus infects human cells there is disruption of rRNA biogenesis. This report examines the effect of adenovirus infection on the nucleolar protein, upstream binding factor (UBF) which plays a major role in regulating rRNA synthesis. We determined that early after infection, UBF associates with the replication of viral DNA, preferentially associating with the ends of the linear viral genome, and that addition of anti-UBF serum to in vitro replication assays markedly reduced viral DNA replication. Regions of UBF important to these observations are also established. Interestingly, sequestering the majority of UBF from the nucleolus did not lead to the ablation of rRNA synthesis or the sequestration of RNA pol I. In infected cells the bulk of RNA synthesis was RNA pol I associated and distinct from the location of most of the detectable UBF. We propose that UBF plays a role in viral DNA replication, further strengthening the role of nucleolar antigens in the adenovirus life cycle.Journal of Cell Science 07/2006; 119(Pt 12):2621-31. · 6.11 Impact Factor -
Article: Nucleolar dominance: a model for rRNA gene silencing.
Genes & Development 06/2006; 20(10):1207-14. · 11.66 Impact Factor -
Article: A role for upstream binding factor in organizing ribosomal gene chromatin.
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ABSTRACT: Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.Biochemical Society Symposium 02/2006; · 2.74 Impact Factor -
Article: UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery.
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ABSTRACT: Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.Genes & Development 02/2005; 19(1):50-64. · 11.66 Impact Factor -
Article: Oddpols united: new insights into transcription by RNA polymerases I and III.
Gene Expression 02/2002; 10(5-6):263-9. · 1.31 Impact Factor -
Article: UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat.
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ABSTRACT: The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.Molecular and Cellular Biology 02/2002; 22(2):657-68. · 5.53 Impact Factor -
Article: Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli
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ABSTRACT: Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.The EMBO Journal 06/2001; · 9.20 Impact Factor -
Article: DNasel-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA
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ABSTRACT: We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X.laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAasel. Unlike DNAasel, Endo R. Mspl digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this “proximal domain” is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function. -
Article: On the formation of amplified nucleoli during early Xenopus oogenesis.
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ABSTRACT: In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF's role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.Journal of Structural Biology 140(1-3):214-26. · 3.41 Impact Factor
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2001–2008
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University of Dundee
Dundee, SCT, United Kingdom
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