GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

Starešinčić, Lidija; Walker, Jane; Dirac-Svejstrup, A. Barbara; Mitter, Richard; Svejstrup, Jesper Q.

doi:10.1074/jbc.m111.286161

Cited by 38 publications

(64 citation statements)

References 32 publications

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“…In addition, a single Q110L mutation, predicted to disrupt buttressing of the GPN loop by residue Q110 in motif G3, was also inactive. All mutated residues shown here to be involved in catalysis in vitro are essential in vivo (7,21). Furthermore, purified Npa3 formed a dimer, and in the Npa3-GDP crystals, two symmetry-related complexes formed a dimer that contained the GPN loop of one monomer in the active site of the other monomer, as observed for the archaeal structure.…”

Section: Resultsmentioning

confidence: 64%

“…Depletion of human GPN3 (20) or mutation of yeast GPN2 or GPN3 (12) also leads to the cytoplasmic accumulation of Rpb1, indicating a general role of all three GPN-loop GTPases in Pol II biogenesis. Depletion of the GPN1 homolog Npa3 from the yeast Saccharomyces cerevisiae leads to the cytoplasmic accumulation of Rpb1 and Rpb3 (21). Rpb1 accumulation is also observed when Npa3 is mutated in its nucleotide-binding site or GPN motif (7,21).…”

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confidence: 89%

“…Depletion of the GPN1 homolog Npa3 from the yeast Saccharomyces cerevisiae leads to the cytoplasmic accumulation of Rpb1 and Rpb3 (21). Rpb1 accumulation is also observed when Npa3 is mutated in its nucleotide-binding site or GPN motif (7,21). The association of yeast Npa3 with Rpb1 is regulated by GTP binding in whole-cell extracts (21), and a direct interaction of human GPN1 and GPN3 with the recombinant Pol II subunits Rpb4 and Rpb7 and the C-terminal repeat domain (CTD) of Rpb1 has been reported (11).…”

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confidence: 99%

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Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Niesser

Wagner

Kostrewa

et al. 2016

Molecular and Cellular Biology

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bBiogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

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Section: Resultsmentioning

confidence: 64%

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confidence: 89%

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confidence: 99%

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Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Niesser

Wagner

Kostrewa

et al. 2016

Molecular and Cellular Biology

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“…Assembly of the RNA polymerases in both humans and yeast is proposed to occur in the cytoplasm as a prerequisite for their nuclear import with the action of several factors (23,30,32,(60)(61)(62). However, little is known about the disassembly, degradation, and recycling of the RNA polymerases.…”

Section: Discussionmentioning

confidence: 99%

“…Rpb2/Rpb3 remain associated, and Rpb6 may associate with this intermediary, pointing to the action of an assembly factor, which could be the prefoldin Bud27. Reassociation of different subunits in the cytoplasm with newly synthesized Rpb1 occurs sequentially, involving different assembly and transport factors (30,32,(60)(61)(62); it is suggested that Bud27 acts in a final step for correct assembly of Rpb5 (23). Whole RNA pol II associated with assembly and transport factors enters the nucleus, and these shuttle it again to the cytoplasm, some of them by a Crm1-dependent mechanism, Bud27 by a Crm1-independent mechanism (23).…”

Section: Fig 10mentioning

confidence: 99%

Correct Assembly of RNA Polymerase II Depends on the Foot Domain and Is Required for Multiple Steps of Transcription in Saccharomyces cerevisiae

Garrido-Godino

García-López

Navarro

2013

Molecular and Cellular Biology

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bRecent papers have provided insight into the cytoplasmic assembly of RNA polymerase II (RNA pol II) and its transport to the nucleus. However, little is known about the mechanisms governing its nuclear assembly, stability, degradation, and recycling. We demonstrate that the foot of RNA pol II is crucial for the assembly and stability of the complex, by ensuring the correct association of Rpb1 with Rpb6 and of the dimer Rpb4-Rpb7 (Rpb4/7). Mutations at the foot affect the assembly and stability of the enzyme, a defect that is offset by RPB6 overexpression, in coordination with Rpb1 degradation by an Asr1-independent mechanism. Correct assembly is a prerequisite for the proper maintenance of several transcription steps. In fact, assembly defects alter transcriptional activity and the amount of enzyme associated with the genes, affect C-terminal domain (CTD) phosphorylation, interfere with the mRNA-capping machinery, and possibly increase the amount of stalled RNA pol II. In addition, our data show that TATA-binding protein (TBP) occupancy does not correlate with RNA pol II occupancy or transcriptional activity, suggesting a functional relationship between assembly, Mediator, and preinitiation complex (PIC) stability. Finally, our data help clarify the mechanisms governing the assembly and stability of RNA pol II. RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs but contributes less than 10% of the total RNA present in growing cells (1). It consists of 12 protein subunits with a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). The catalytic core of the bacterial and eukaryotic enzymes is highly conserved through evolution. However, only five subunits have bacterial homologs (Rpb1, Rpb2, Rpb3, Rpb6, and Rpb11); the others are common to archaea but have no eubacterial homologs (2, 3). The RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides, including general transcription factors, coregulators, and specific transcription activators as well as repressors (1).Many studies have contributed to the knowledge of physical interactions between RNA pol II and transcriptional regulators and have enabled the identification of regions that are important for transcription, from initiation to mRNA export (2, 4-12). In addition, we have recently reported the existence of five "conserved domains," located at the surface of the structure of the complex, with poor or no conservation in their paralogs in RNA polymerases I (Rpa190 and Rpa135) and III (Rpc160 and Rpc128) and in their homologs in archaea and bacteria and demonstrate that all of them make contact with transcriptional regulators (10).One of these regions corresponds to the foot domain (2, 10), which, in cooperation with the "lower jaw," the "assembly" domain, and the "cleft" regions, constitutes the "shelf" module of RNA pol II, which might contribute to the rotation of the DNA as it advances toward the active center (2,8). This domain, conserv...

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Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

et al. 2017

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Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation.

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GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

Cited by 38 publications

References 32 publications

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

Correct Assembly of RNA Polymerase II Depends on the Foot Domain and Is Required for Multiple Steps of Transcription in Saccharomyces cerevisiae

Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1‐inhibitable manner

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