A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export

Reyes-Pardo, Humberto; Barbosa-Camacho, Angel A.; Pérez-Mejía, Ana E.; Lara‐Chacón, Bárbara; Robledo-Rivera, Angélica Y.; Montero-Morán, Gabriela M.; Lara-González, Samuel; Calera, Mónica R.; Sánchez‐Olea, Roberto

doi:10.1016/j.bbamcr.2012.07.001

Cited by 21 publications

(17 citation statements)

References 36 publications

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“…To our surprise, the results indicate that GPN1/RPAP4 silencing induces nuclear retention of RPAP2 as determined by western blotting after cell fractionation (Figure 5C). Together with our data on binding and localization domains, this result indicates that RPAP2 returns to the cytoplasm in association with the GTPase GPN1/RPAP4, whose nuclear export in CRM1 dependent (39). …”

Section: Resultssupporting

confidence: 83%

“…Nuclear import of pre-assembled RNAP II requires at least two specific factors, GPN1/RPAP4 and RPAP2. The GTPase GPN1/RPAP4 shuttles between the cytoplasm and the nucleus through the action of a classical NES (39). RPAP2 is imported to the nucleus in association with RNAP II, an association that requires an RPAP2 domain encompassing its first 170 residues.…”

Section: Discussionmentioning

confidence: 99%

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Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

Forget¹,

Lacombe²,

Cloutier³

et al. 2013

Nucleic Acids Research

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The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1–170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156–612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.

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Section: Resultssupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

Forget¹,

Lacombe²,

Cloutier³

et al. 2013

Nucleic Acids Research

View full text Add to dashboard Cite

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“…Homo-and heterodimerization of GPN1 and its paralogs were reported previously (10)(11)(12). Npa3 contains a nuclear export sequence (NES) (residues 286 to 295) (13), consistent with the predominant cytoplasmic localization of Npa3 in yeast (14,15) and GPN1 in human cells (7,13,16,17).…”

mentioning

confidence: 56%

“…Our model does not include any nuclear roles of Npa3, although they may exist, because nucleocytoplasmic shuttling of GPN1/Npa3 was reported previously (7,13,21,52). However, GPN-loop GTPases lack a nuclear localization signal (NLS), and mutations of GPN2 or GPN3 cannot be rescued by the fusion of a NLS to Rpb3 (12), consistent with the main function of these enzymes being cytosolic.…”

Section: Figmentioning

confidence: 95%

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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“…Thus, multiple interactions between GPN1/RPAP4 (Npa3p in yeast) and RNA pol II subunits in the soluble fraction of human cell extracts have been identified by performing protein affinity purification coupled to mass spectrometry (AP-MS) (5). GPN1 is a cytoplasmic-nuclear shuttling GTPase (5,6) that associates with RNA pol II in a GTP-dependent manner (7). The involvement of GPN1 and its Saccharomyces cerevisiae homolog, Npa3p, in the nuclear import of RNA pol II has been confirmed by other reports (7,8).…”

mentioning

confidence: 99%

Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

Gómez-Navarro

Peiró-Chova

Rodrı́guez-Navarro

et al. 2013

Molecular and Cellular Biology

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The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex. In eukaryotes, RNA polymerase II (RNA pol II) synthesizes mRNA and several noncoding RNAs. RNA pol II is a multisubunit enzyme composed of 12 subunits that are highly conserved among eukaryotes and whose structure has been well characterized (1). Yeast RNA pol II can dissociate into a 10-subunit catalytic core and a heterodimer of subunits Rpb4p and Rpb7p, which are required for the initiation from promoter DNA (2). The two large subunits, Rpb1p and Rpb2p, form the central mass of the enzyme, whereas the smaller subunits generally bind on the surface. Although the structure and functional regulation of RNA pol II have been characterized in detail, the study of its biogenesis has only recently been pursued. By assuming that the assembly of eukaryotic RNA pol II is similar to that of the bacterial polymerase, as subunit dissociation experiments suggest (3), it has been proposed that RNA pol II assembly would start with the formation of the Rpb3 subassembly, followed by the docking of the Rpb2 subassembly and finally the binding of the Rpb1 subassembly (4). The assembly process requires the participation of proteins that are not components of the mature enzyme. Several proteins that interact transiently with RNA pol II with a putative role as assembly factors have been identified. Thus, multiple interactions between GPN1/RPAP4 (Npa3p in yeast) and RNA pol II subunits in the soluble fraction of human cell extracts have been identified by performing protein affinity purification coupled to mass spectrometry (AP-MS) (5). GPN1 is a cytoplasmic-nuclear shuttling GTPase (5, 6) that associates with RNA pol II in a GTP-dependent manner (7). The involvement of GPN1 and its Saccharomyces cerevisiae homolog, Npa3p, in the nuclear import of RNA pol II has been confirmed by other reports (7, 8). Using MS-based quantitative proteomics on human cell extracts, a cytoplasmic RNA pol II intermediate containing the Rpb1p and Rpb8p subunits associated with th...

show abstract

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export

Cited by 21 publications

References 36 publications

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

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

Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

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