Treacle recruits RNA polymerase I complex to the nucleolus that is independent of UBF

Lin, Chen-I; Yeh, Ning-Hsing

doi:10.1016/j.bbrc.2009.06.050

Cited by 54 publications

(56 citation statements)

References 26 publications

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“…We therefore used the coefficient of variation (the standard deviation normalized to the mean) of nucleolin voxel intensities within 3D reconstructions of DAPI-defined nuclei as a metric for nucleolin aggregation. Indeed, destabilization of DD-TCOF1 appears to result in reduced nucleolin aggregation (Figure 4B), reminiscent of previous findings that TCOF1 knockdown results in dispersion of Pol I and UBF away from nucleoli [11]. Since it is possible that a mutation specific to clone 1 was able to modulate the functional consequence of TCOF1 destabilization, especially in light of possible off-target effects of the CRISPR/Cas9 system [26]–[31], we confirmed that independent clone 2 also showed destabilization of TCOF1 and significantly reduced nucleolin aggregation (p<0.0001) in the absence of Shield-1 (Figure S4).…”

Section: Resultssupporting

confidence: 81%

“…Although siRNA-mediated knockdown of TCOF1 has been attempted in the past, protein reduction is significantly apparent after four days [10], [11]; the long time required to achieve significant knockdown may allow secondary effects of reduced viability to accumulate, which might confound the interpretation of results. Our present work demonstrates that efficient complete knock-in of DD-tagged TCOF1 via the CRIPSR/Cas9 system enables relatively rapid knockdown of protein levels upon removal of the stabilizing Shield-1 compound, or conversely, rapid upregulation of TCOF1 levels upon addition of Shield-1.…”

Section: Discussionmentioning

confidence: 99%

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CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

et al. 2014

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Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles.The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

et al. 2014

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“…Nucleolar mis-localization phenotypes have also been observed in HeLa cells treated with actinomycin D, an inhibitor of RNA Pol I [56], which is a TCOF1/Treacle interacting protein [57]. It is possible that WDR43 may also function together with TCOF1 and Nopp140 to recruit proteins to the nucleolar organizer regions (NORs) and the upstream binding factor (UBF), an RNA PolI transactivator [19].…”

Section: Discussionmentioning

confidence: 99%

Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

et al. 2014

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During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

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“…TCS presents as a collection of craniofacial malformations that arise during early development 131 , 132 . Treacle is similar to Nopp140 in motif composition and likely function; 133 , 134 both proteins are considered chaperones for nucleolar snoRNPs, however, treacle (and perhaps Nopp140) may also play a vital role in recruiting UBF and Pol I to the rDNA promoter 135 . Haplo-insufficiencies in treacle ( Tcof +/− ) cause a block in 2’- O -ribose methylation, 136 an overall loss of cytoplasmic ribosomes, 137 and most telling, a loss of specialized embryonic neural crest cells that ostensibly have a high demand for protein synthesis and thus functional ribosomes 131 .…”

Section: Endogenous Mutations: the Ribosomopathiesmentioning

confidence: 99%

Nucleolar stress with and without p53

James

Wang

Raje

et al. 2014

Nucleus

238

280

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A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell’s energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53?

show abstract

Treacle recruits RNA polymerase I complex to the nucleolus that is independent of UBF

Cited by 54 publications

References 26 publications

CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

Nucleolar stress with and without p53

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