RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors 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 high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.

Over the past few years, the study of protein-protein interactions and protein complexes has shed more light on cellular processes and cell function. Because alterations in protein-protein interactions perturb the normal sequence of events in the cell and contribute to diseases such as cancer, the understanding of both the normal cellular protein-protein interaction networks and their modulation during the establishment of disease is a crucial issue in biomedical research, as it will facilitate the development of drugs to fight these diseases. In this article, the most commonly used approaches for studying protein-protein interactions are discussed as well as the direction in which the field of systematic characterization of protein interaction networks is progressing. We also discuss some success stories in the modulation of disease-related protein-protein interactions using small molecules.

Over the past 3 decades, many efforts have been made to identify and characterize the factors that regulate the activity of RNA polymerase II (RNAPII), the enzyme that synthesizes all the mRNA and many small nuclear RNA in eukaryotes. Quite surprisingly, very little is known about the cell machinery that regulates the fate of RNAPII before or after transcription. In this study, we report that XAB1/GPN1, a member of a unique class of GTPases containing a conserved GPN‐loop, is involved in the biogenesis of RNAPII by regulating its nuclear import. XAB1/GPN1 is physically associated with RNAPII in the soluble cell fraction and is essential for cell growth. Accumulation of RNAPII in the cytoplasm of the cell was induced upon (i) depletion of XAB1/GPN1 by siRNA silencing, (ii) overexpression of a XAB1‐GFP dominant negative fusion protein, (iii) mutation of the GTP‐binding or GPN motifs of the XAB1/GPN1 homologue in yeast, NPA3, and (iv) treatment of cells with leptomycin B, an inhibitor of XPO1/CRM1‐dependent nuclear export. Yeast strains with mutations in the NPA3 gene showed hypersensitivity to benomyl, a specific inhibitor of microtubule assembly, suggesting a role for microtubules in RNAPII biogenesis. Together, these results indicate that XAB1/GPN1 shuttles between the cytoplasm and the nucleus to mediate nuclear import of RNAPII.

We performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Many identified interaction partners were targeted in reciprocal tagging experiments in order to confirm some interactions and to enrich the dataset. High‐confidence interactions were selected computationally using an algorithm that we developed and trained using machine learning to minimize the rate of both false‐positives and false‐negatives. The data produced with 100 affinity tagged proteins was used to (1) build a high‐definition map of interactions that connect components of the transcription and RNA processing machineries in human cells; (2) show that transcription and RNA processing factors from the soluble cellular fraction are associated with proteins that specifically regulate the formation (e.g. assembly, localization and/or stability) of protein complexes; and, (3) assign a putative function to a number of previously‐uncharacterized proteins on a ‘‘guilt by association’’ basis. A number of previously‐uncharacterized proteins that we further characterized functionally and biochemically define a novel class of regulatory factors that target RNA polymerase II and other transcription factors prior and/or after the transcription reaction on chromatin DNA.