The Protein Interaction Network of the Human Transcription Machinery Reveals a Role for the Conserved GTPase RPAP4/GPN1 and Microtubule Assembly in Nuclear Import and Biogenesis of RNA Polymerase II

Forget, Diane; Lacombe, Andrée-Anne; Cloutier, Philippe; Al-Khoury, Racha; Bouchard, André; Lavallée‐Adam, Mathieu; Faubert, Denis; Jeronimo, Célia; Blanchette, Mathieu; Coulombe, Benoit

doi:10.1074/mcp.m110.003616

Cited by 92 publications

(162 citation statements)

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“…Recent observations in yeast and humans are consistent with RNA pol assembly in the cytoplasm as a prerequisite for its nuclear import (23,(30)(31)(32). Thus, our results do not eliminate the possibility that RNA pol II foot mutations affect the correct assembly of Rpb1 with the rest of the complex and that, thus, nonassociated Rpb1 could be degraded in the cytoplasm.…”

Section: Resultssupporting

confidence: 73%

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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Section: Resultssupporting

confidence: 73%

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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“…Interactions of the GPN proteins with RNAPII have been documented in two other recent studies (2,6). Interestingly, GPN proteins have also been demonstrated to interact with the CCT complex, which controls the polymerization of tubulins into microtubules (9).…”

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confidence: 95%

Regulating the Shuttling of Eukaryotic RNA Polymerase II

Croce

2011

Molecular and Cellular Biology

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The eukaryotic RNA polymerase II (RNAPII) transcribes most protein-coding RNAs (mRNAs) and the capped noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) (10). In the past 3 decades, our knowledge about the functional role of RNAPII during gene transcription has dramatically advanced (8). However, there are still several open questions regarding the regulatory mechanisms involved in the steps before and after transcription. In particular, how RNAPII is imported into the nucleus remains elusive. In this issue, Carré and Shiekhattar (3) report that the evolutionarily conserved GTPases GPN1 and GPN3 stably associate with and regulate the nuclear import of the human RNAPII. These exciting results now make it possible to begin to dissect the regulatory pathways of the intracellular localization and nuclear import of RNAPII.Recently, the structures and subunit compositions of the three RNA polymerases (Table 1) have been characterized (4). RNAPII is a highly conserved enzyme composed of 12 subunits, 10 of which form the central core. The other two subunits (Rpb4 and Rpb7) form a peripheral heterodimer that protrudes out from the core enzyme structure. This stabilizes the RNAPII complex in clamp-closed conformation, favoring efficient transcription initiation. Rpb10 and Rpb12 are common subunits for all of the eukaryotic RNA polymerases and are implicated in the interaction with the basal transcription factors, such as the TATA box-binding protein (TBP). These interactions lead to the formation of the preinitiation complex (PIC), which contains RNAPII as well as the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. In the past years, numerous coactivators that both facilitate the access of PIC to its target promoters and increase the stability of the complex have been functionally characterized. Recent data (1) indicate that the RNAPII assembly pathway occurs in the cytoplasm and that assembly is coordinated by the consecutive actions of the cochaperone hSpagh and the chaperone Hsp90. The two largest subunits, Rpb1 and Rpb2, are initially preassembled into two separated subcomplexes, together with several other subunits that are either common to all three RNA polymerases or are specific for RNAPII (Fig. 1). It is likely that the two subcomplexes are then assembled together before they enter the nucleus through the nuclear pore complex (NPC). Interestingly, it now seems that access to the nucleus is restricted exclusively to the assembled RNAPII, thus preventing any premature binding of the independent subunits at promoter regions.But the question of how the assembled RNAPII is imported into the nucleus remains. How is this step regulated? Carré and Shiekhattar tackled this question using a combination of cell biology approaches and mass spectrometry of affinity-purified complexes containing tagged cytoplasmic and nuclear RNAPII (3). They identified GPN1 and GPN3 as novel interactors of the RNAPII, which remained associated with...

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“…For this purpose, we like combining the use of HEK293 cells, for which we and others have already generated significant, although far from complete, protein-protein interaction data, with cell lines specifically originating from the disease/tissue of interest. Mapping interaction networks generally identifies both upstream (i.e., regulators) and downstream (i.e., regulated proteins) effectors of the tagged proteins, as we have shown for components of the transcription machinery [3][4][5]8,15,16,18 and as others have shown for proteins specifically involved in disease conditions. 2,6,10,12,27,32,33 Consequently, given that a protein associated with a specific disease condition represents a putative biomarker for this particular disease, its interactors putatively represent additional biomarkers that can regulate the disease phenotype by acting as upstream or downstream effectors.…”

Section: Biomarker Discoverymentioning

confidence: 99%

“…15 Accordingly, these proteins were named RNAPII-Associated Proteins (RPAPs, namely, RPAP1, 2, 3, and 4). 3,8,15,16 The 77-bait data set (2009) revealed that RPAP3 is part of an 11-subunit protein complex containing POLR2E (RPB5), a subunit shared by all three nuclear RNAPs, and a set of factors previously characterized as being involved in protein complex assembly (according to their Gene Ontology [GO] term). 3 These include the chaperone cofactors RPAP3 and PIH1D1; the classical prefoldins PFDN2 and PFDN6; and the prefoldin-like proteins UXT, PDRG1, and C19orf2/URI.…”

Section: High-resolution Maps Of the Protein Interactome For The Rna mentioning

confidence: 99%

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Mapping the Disease Protein Interactome: Toward a Molecular Medicine GPS to Accelerate Drug and Biomarker Discovery

Coulombe

2011

J. Proteome Res.

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Genomic approaches such as genome-wide association studies (GWAS), disease genome sequencing projects, and genome-wide expression profiling analyses, in conjunction with classical genetic approaches, can identify human genes that are altered in disease, thereby suggesting a role for the encoded protein (or RNA) in the establishment and/or progression of the disease. However, many technical difficulties challenge our ability to validate the role of these disease-associated genes and gene products. Moreover, many identified genes contain open reading frames (ORFs) that have yet to be annotated, that is, the function (or activity) of the encoded protein is unknown. As a result, translating the genomic information available in public databases into useful tools for understanding and curing disease is a very slow and inefficient process. To overcome these difficulties, we have developed a technology platform, termed the "molecular medicine GPS" (mm-GPS), which is aimed at defining high-quality maps of interaction networks involving disease proteins. These maps are used to identify network dysfunctions in disease cells or models and to develop molecular tools such as RNA interference (RNAi) and small-molecule inhibitors to further characterize the molecular basis of disease. In this article, I review our progress in producing high-quality maps of human protein interaction networks, and I describe how we used this information to identify new factors and pathways that regulate the RNA polymerase II transcription machinery. I also describe how we utilize the mm-GPS platform to guide more efficient efforts leading from disease-associated genes to protein interaction networks to smallmolecule inhibitors, and consequently, to accelerate drug and biomarker discovery. KeywordsProtein interaction networks; disease-associated genes; RNA polymerase II; transcription factors; biomarker and drug discovery; technology platform Interaction Networks As a New, Systems-Based "Descriptor" of Proteins CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptof the previously characterized interactors of a protein will inform on its function and localization within the cell. In addition, the network of interactors of a protein will often include regulatory factors that participate in modulating its activity. The value of this information, which must be validated using appropriate functional and biochemical assays, will increase with the quality of the interaction map.A number of experimental methods (see Figure 1 for Novel approaches, such as LUminescence-based Mammalian IntERactome mapping (LUMIER), 1 protein-fragment complementation assay (PCA), 20 and high throughput imaging of protein localization 29 have also been developed to help map protein-protein interactions in space and time in mammalian cells (see Figure 1 for a comparative description). Although these approaches have not been widely used to date, they will most certainly serve to enhance the confidence of protein-protein interactions by helping to desc...

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The Protein Interaction Network of the Human Transcription Machinery Reveals a Role for the Conserved GTPase RPAP4/GPN1 and Microtubule Assembly in Nuclear Import and Biogenesis of RNA Polymerase II

Cited by 92 publications

References 52 publications

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

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

Regulating the Shuttling of Eukaryotic RNA Polymerase II

Mapping the Disease Protein Interactome: Toward a Molecular Medicine GPS to Accelerate Drug and Biomarker Discovery

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