An Eukaryotic RuvB-like Protein (RUVBL1) Essential for Growth

Qiu, Xiao‐Bo; Lin, Yi-Ling; Thome, Kelly C.; Pian, Phillip; Schlegel, Brian P.; Weremowicz, Stanislawa; Parvin, Jeffrey D.; Dutta, Anindya

doi:10.1074/jbc.273.43.27786

Cited by 128 publications

(156 citation statements)

References 41 publications

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“…These multiple interactions of Pontin/Reptin suggest an essential physiological role, and studies in S. Cerevisiae have demonstrated that yeast orthologs of both Reptin and Pontin are required for cell viability without overall shutdown of transcription. 7,8 Essential deveopmental roles in Drosophila and Xenopus have also been observed. 9,10 However, the only information currently available about their role in mammalian cells is that knockdown of Pontin in human diploid fibroblasts causes proliferation arrest, 11 and that knockdown of Ruvbl1 (and other Tip60 complex components) in embryonic stem (ES) cells induces loss of pluripotency.…”

Section: Introductionmentioning

confidence: 92%

Pontin is essential for murine hematopoietic stem cell survival

et al. 2012

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© F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o nMethods). Western blotting was performed on cell/tissue extracts (Online Supplementary Appendix) using Tip48, Tip49 19, PCNA (Sigma) and β-tubulin (Sigma) antibodies as described. 20 Results and DiscussionTo address the physiological role of Pontin we first generated a Ruvbl1 null allele by targeting the mouse germline (Online Supplementary Figure S1). Intercrossing of Ruvbl1 +/-mice did not generate any viable offspring, and no postimplantation Ruvbl1 -/-embryos could be retrieved ( Figure 1A). A few Ruvbl1 -/-blastocysts were identified. However, upon culturing, which results in outgrowth of the pluripotent inner cell mass, no proliferating cultures were observed to be Ruvbl1 -/-. We conclude from this that Pontin is required for embryogenesis at a very early stage, possibly involving the proliferation of pluripotent inner mass cells.To address the role of Pontin after development, we analyzed the Pontin and Reptin expression patterns in the adult mouse and selected cell lines. Overall, the expression of Pontin, but not of Reptin, correlated with that of proliferating cell nuclear antigen (PCNA), consistent with Pontin playing a specific role in cell proliferation. In particular, we found Pontin to be highly expressed in hematopoietic tissues (bone marrow, spleen, thymus, lymph nodes), as well as pluripotent cells/tissues (ES cells, testis), with low levels in liver, brain and lung ( Figure 1B). To address Pontin function in the hematopoietic system, a conditional Ruvbl1 allele (Online Supplementary Figure S1) © F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o ndays post-injection, surviving Pontin cKO mice displayed a massive loss of bone marrow cellularity ( Figure 1E). This involved the coordinated loss of myeloid and lymphoid cells ( Figure 1E) and was preceeded by the complete loss of Lin -Sca-1 + c-Kit + (LSK) HSCs ( Figure 1F). To determine whether the observed effects could be attributed to an intrinsic hematopoietic requirement for Pontin, we transplanted CD45.2 Pontin cKO and Pontin Con bone marrow noncompetitively into irradiated CD45.1/2 recipients. Subsequent polyIC induction resulted in loss of hematopoietic cells ( Figure 1G and H) and lethality ( Figure 1I) Figure 2B-E), and a very high proportion of the remaining Pontin cKO HSCs were apoptotic, as measured by staining for AnnexinV and intracellular DNA ( Figure 2F). From this we conclude that Pontin is required for HSC viability and that in its absence HSCs undergo apoptosis.These results demonstrate an essential role for Pontin in early mammalian development, consistent with its presence in multiple complexes carrying out essential cellular functions. In addition, the definitive hematopoietic system in general, and HSCs in particular, were critically dependent on Pontin for its maintenance, with HSC apoptosis and depletion being an immediate consequence of Ruvbl1 inactivation. This is in contras...

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Section: Introductionmentioning

confidence: 92%

Pontin is essential for murine hematopoietic stem cell survival

et al. 2012

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“…The R2TP module RUVBL1 and RUVBL2 (RVB1 and RVB2; TIP49 and TIP48; pontin and reptin) RUVBL1 and RUVBL2 are ATPases of the AAA+ superfamily (ATPase associated with diverse cellular activities) and are related to the prokaryotic DNA helicase RuvB (Qiu et al 1998;Kanemaki et al 1999), which is involved in homologous recombination and doublestrand break repair (see Fig. 2 for a schematic representation of the R2TP module components).…”

Section: Introductionmentioning

confidence: 99%

New insights into the biogenesis of nuclear RNA polymerases?This paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Cloutier

Coulombe

2010

Biochem. Cell Biol.

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More than 30 years of research on nuclear RNA polymerases (RNAP I, II, and III) has uncovered numerous factors that regulate the activity of these enzymes during the transcription reaction. However, very little is known about the machinery that regulates the fate of RNAPs before or after transcription. In particular, the mechanisms of biogenesis of the 3 nuclear RNAPs, which comprise both common and specific subunits, remains mostly uncharacterized and the proteins involved are yet to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by nuclear RNAP subunits from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a highconfidence dataset and uncovered novel interactors that regulate the RNAP II transcription machinery, including a set of proteins we named the RNAP II-associated proteins (RPAPs). One of the RPAPs, RPAP3, is part of an 11-subunit complex we termed the RPAP3/R2TP/prefoldin-like complex. Here, we review the literature on the subunits of this complex, which points to a role in nuclear RNAP biogenesis.

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“…Because TIH1 had been shown to be an essential gene (9,19,22), we first asked if TIH2 is also required for viability. The TIH2 gene was disrupted as described under "Experimental Procedures."…”

Section: Tih2mentioning

confidence: 99%

“…The recombinant TIP49a is an ATP-dependent DNA helicase (18). Meanwhile, a human homologue of p50, RUVBL1, was shown to co-purify with RNAP II holoenzyme, and the yeast counterpart was reported to be essential for viability (19). Furthermore, in a search for proteins that interact with ␤-catenin, two novel human proteins, 52-and 44-kDa, were detected (20).…”

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confidence: 99%

The Saccharomyces cerevisiae RuvB-like Protein, Tih2p, Is Required for Cell Cycle Progression and RNA Polymerase II-directed Transcription

Lim¹,

Kimata²,

Ohdate³

et al. 2000

Journal of Biological Chemistry

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Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant, tih2-160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G 1 phase. Northern blot analysis revealed that Tih2p is required for transcription of G 1 cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2-160 and those of taf145 mutants suggest a role for TIH2 in the regulation of RNA polymerase II-directed transcription.One of the common post-translational modifications of nuclear and cytosolic proteins in eukaryotes is the addition of N-acetylglucosamine residues O-linked (O-GlcNAc) 1 to serine and threonine residues. A number of physiologically or structurally important proteins have thus far been shown to contain O-GlcNAc, including the largest subunit of RNA polymerase II (RNAP II) (1), transcription factors such as Sp1 and hepatocyte nuclear factor 1 (2-4), nuclear pore proteins (5, 6) and chromatin-associated proteins (7). To study nuclear factors involved in nuclear transport and other nuclear events, we previously performed an in vitro binding assay of a rat liver nuclear matrix fraction using a wheat germ agglutinin affinity column (8).Several O-GlcNAc-containing proteins such as nucleoporins as well as nonglycosylated proteins like importin ␤ were isolated (9). Two RuvB-like proteins, p50 (9) and p47 (10), were also isolated by this method probably because of their interaction with O-GlcNAc-bearing proteins. RuvB is a prokaryotic DNA helicase, and its helicase activity and DNA binding affinity are enhanced by interaction with RuvA (11,12). These two factors form a large motor protein complex to promote branch migration at Holliday junctions at the late stages of homologous recombination. The p50/p47 and RuvB proteins share highly conserved Walker domains (A and B), indicative of proteins that bind nucleotide triphosphates (10, 13). Based on this, p50 and p47 were proposed to be the eukaryotic homologues of the RuvB DNA helicase. Indeed, p50 and p47 are present in a wide range of eukaryotes ranging from yeast to mammals, suggesting that this basic helicase activity may be conserved among the eukaryotes.In addition to recombination, transient unwinding of the DNA duplex is required in many cellular processes such as replication, transcription, and DNA repair. To accommodate ...

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An Eukaryotic RuvB-like Protein (RUVBL1) Essential for Growth

Cited by 128 publications

References 41 publications

Pontin is essential for murine hematopoietic stem cell survival

Pontin is essential for murine hematopoietic stem cell survival

The Saccharomyces cerevisiae RuvB-like Protein, Tih2p, Is Required for Cell Cycle Progression and RNA Polymerase II-directed Transcription

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