Yeast Nuclear Extract Contains Two Major Forms of RNA Polymerase II Mediator Complexes

Liu, Ying; Ranish, Jeffrey A.; Aebersold, Ruedi; Hahn, Steven

doi:10.1074/jbc.m009586200

Cited by 178 publications

(112 citation statements)

References 67 publications

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“…After fixation in 3.7% formaldehyde for 20 min, immunofluorescence microscopy on yeast spheroplasts was performed as previously described (Pemberton et al, 1997). Anti-Sua7 rabbit polyclonal antibodies were used to detect Sua7p (Liu et al, 2001; a gift from David Auble, University of Virginia), followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson Laboratories, ImmunoResearch Laboratories, West Grove, PA). Cells were mounted in Hoechst stain solution (bisbenzimide).…”

Section: Cell Culture and Microscopymentioning

confidence: 99%

Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

Hodges

Leslie

Mosammaparast

et al. 2005

MBoC

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Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes. INTRODUCTIONIn eukaryotic cells the nucleocytoplasmic transport of most proteins and some RNAs is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (also referred to as importins and exportins; reviewed in Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). After synthesis in the cytoplasm, most nuclear protein cargoes are bound by a member of the karyopherin family, through direct interaction with a nuclear localization sequence contained in the cargo protein. Transport through the nuclear pore complex occurs via transient interactions of the karyopherin with the NPC. Once in the nucleus, the karyopherin encounters a high concentration of RanGTP, which acts as a regulator of transport. Interaction of the karyopherin with RanGTP leads to dissociation of the karyopherin from its nuclear cargo (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In some circumstances, nuclear-binding partners of the cargo appear to also play a role in stimulating the dissociation of Kap and cargo (Senger et al., 1998;Lee and Aitchison, 1999;Pemberton et al., 1999). In yeast, there are 14 members of the karyopherin family, with Ͼ20 members in mammalian cells (Mosammaparast and Pemberton, 2004). Karyopherins appear to function in either nuclear import or nuclear export, with only two examples of a Kap that works in both directions (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In yeast, 11 members of the karyopherin family must import at least 1500 nuclear proteins, suggesting that each receptor must have many cargoes. To date specific transport receptor-cargo pairs have only been shown for ϳ30 cargoes; in addition the NLS sequences recognized by most Kaps is n...

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Section: Cell Culture and Microscopymentioning

confidence: 99%

Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

Hodges

Leslie

Mosammaparast

et al. 2005

MBoC

View full text Add to dashboard Cite

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“…The two major MMPs, that is MMP‐1 and MMP‐13, cleave full‐length CTGF into the N‐terminal and C‐terminal fragments of similar molecular weights 19, 24. Due to the absence of MMP‐1 in adult mice 25, 26, MMP‐13 is primarily responsible for the cleavage of full‐length CTGF into its fragments that are more potent to induce hepatic fibrogenesis, as it has been reported that the N‐terminal fragment of CTGF mediates myofibroblast differentiation and collagen synthesis and the C‐terminal fragment stimulates fibroblast proliferation 27. The N‐terminal fragment also acts as downstream mediator of TGF‐β1 and increases in fibrotic processes 27, 28.…”

Section: Introductionmentioning

confidence: 99%

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

George

Tsutsumi

Tsuchishima

2017

J Cellular Molecular Medi

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Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.

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“…[25][26][27] Two major forms of Mediator complexes have been identified by mass spectroscopy. These consist of a core complex with or without association of the Cdk8 module, 28,29 which comprises the 4 proteins Cdk8, Med13, Ccnc and Med12. [30][31][32][33] The Cdk8 module or RNA Polymerase II (Pol II) bind mutually exclusive to Mediator, which likely constitutes a means of regulating Mediator function.…”

Section: Introductionmentioning

confidence: 99%

Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation

et al. 2016

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Mediator is considered an enhancer of RNA-Polymerase II dependent transcription but its function and regulation in pluripotent mouse embryonic stem cells (mESCs) remains unresolved. One means of controlling the function of Mediator is provided by the binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to the core Mediator. Here we report that Med12 operates together with PRC1 to silence key developmental genes in pluripotency. At the molecular level, while PRC1 represses genes it is also required to assemble ncRNA containing Med12-Mediator complexes. In the course of cellular differentiation the H2A ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the association of Cdk8 with Mediator. This remodeling of Mediator-associated protein complexes converts Mediator from a transcriptional repressor to a transcriptional enhancer, which then mediates ncRNA-dependent activation of Polycomb target genes. Altogether, our data reveal how the interplay of PRC1, ncRNA and Mediator complexes controls pluripotency and cellular differentiation.

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Yeast Nuclear Extract Contains Two Major Forms of RNA Polymerase II Mediator Complexes

Cited by 178 publications

References 67 publications

Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation

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