[Show abstract][Hide abstract] ABSTRACT: Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Here, we investigate the biogenesis and propagation of nucleoli, sites of ribosome biogenesis and key regulators of cellular growth. Nucleolar and cell cycles are intimately connected. Nucleoli disappear during mitosis, reforming around prominent uncharacterized chromosomal features, nucleolar organizer regions (NORs). By examining the effects of UBF depletion on both endogenous NORs and synthetic pseudo-NORs, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Furthermore, we demonstrate that neo-NORs, UBF-binding site arrays coupled with rDNA transcription units, direct the de novo biogenesis of functional compartmentalized neonucleoli irrespective of their site of chromosomal integration. For the first time, we establish the sequence requirements for nucleolar biogenesis and provide proof that this is a staged process where UBF-dependent mitotic bookmarking precedes function-dependent nucleolar assembly.
Genes & development 01/2014; · 12.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of ∼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.
Nucleic Acids Research 09/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The short-arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms, and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleolar organizer regions (NORs) are comprised of tandem arrays of ribosomal gene repeats. During interphase ribosomal genes are transcribed by RNA Polymerase I resulting in the formation of a nucleolus. Within nucleoli an intricate and highly coordinated assembly pathway is responsible for the production of biology’s most complex machine, the ribosome. Upstream Binding Factor (UBF) binds extensively over and plays a key role in organizing ribosomal gene chromatin throughout the cell cycle. It is responsible for the appearance of active NORs as secondary constrictions on metaphase chromosomes and its levels determine the proportion of ribosomal gene repeats that are active in a given cell type. Extensive UBF binding to NORs directs recruitment of many factors required in the early steps of ribosome biogenesis, thus enabling efficient nucleolar reformation. Finally we reveal that UBF, once thought to be restricted to vertebrates, is present in many animal phyla.
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. · 17.98 Impact Factor
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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. · 12.44 Impact Factor
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] ABSTRACT: The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.
Biochemical Society Transactions 01/2006; 33(Pt 6):1441-3. · 2.59 Impact Factor
[Show abstract][Hide abstract] 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. · 12.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An asylum would seem to be a fitting place to assemble a group that fondly refers to itself as “oddpols,” and indeed it is, once every 2 years. The most recent gathering was officially billed as the Third International Conference on Transcription by RNA Polymerases I and III, which took place once again at the Asilomar (“asylum by the sea”) Conference Grounds in Pacific Grove, CA, June 5–9, 2002. Of course, asylum has multiple meanings, and Asilomar fits the more pleasant definition of “sanctuary.” There among the dunes and windswept Monterey Pines, a dedicated and lively group of principal investigators, postdocs, and graduate students shared their insights into the workings of the two RNA polymerases responsible for most of the RNA synthesis that occurs in a eukaryotic nucleus. The meeting, organized by Marvin Paule (Colorado State University), Ian Willis (Albert Einstein College of Medicine), and Craig Pikaard (Washington University, St. Louis), was an international affair, bringing together leading research labs from India, Germany, France, Italy, Australia, Canada, Puerto Rico, the United Kingdom, and the United States.
[Show abstract][Hide abstract] 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. · 5.37 Impact Factor
[Show abstract][Hide abstract] 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 01/2002; 140(1-3):214-26. · 3.36 Impact Factor
[Show abstract][Hide abstract] 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.