Diane Forget

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

ABSTRACT We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

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.

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.

High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes

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.

Photo-Cross-Linking of a Purified Preinitiation Complex Reveals Central Roles for the RNA Polymerase II Mobile Clamp and TFIIE in Initiation Mechanisms

ABSTRACT The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIEα closely approaches promoter DNA at nucleotide −10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIEα is obtained when the DNA template is artificially premelted in the −11/−1 region, suggesting that TFIIEα facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription.

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.

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

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.

Discovery of Cell Compartment Specific Protein–Protein Interactions using Affinity Purification Combined with Tandem Mass Spectrometry

Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A.

Absence of neurological abnormalities in mice homozygous for the Polr3a G672E hypomyelinating leukodystrophy mutation

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.

Use of site-specific protein-DNA photocrosslinking of purified complexes to analyze the topology of the RNA polymerase II transcription initiation complex.

A method for the photocrosslinking of proteins to DNA in purified complexes is described. It makes use of the juxtaposition of a limited number of photoreactive nucleotides with a limited number of radiolabeled nucleotides at a specific location in a DNA fragment. Protein-DNA complexes are submitted to an electrophoretic mobility shift assay that is then irradiated with UV light in order to crosslink the proteins to DNA. The specific complexes are localized on the gel, purified, and processed for the identification of the crosslinked polypeptides.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z‐disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown.