We have 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. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy.

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position ؊3 to ؉6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position ؊6 to ؉6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position ؊19 to ؉8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.To counteract the detrimental effect of genotoxic attacks, cells activate sophisticated and specific DNA repair pathways. Damage induced by UV radiation, environmental agents, and anticancer drugs are removed by two distinct nucleotide excision repair (NER) 1 subpathways, namely global genome repair (GGR), which eliminates lesions from the entire genome, and transcription-coupled repair (TCR), a specialized pathway that repairs damages on a transcribed strand of active genes (1-3). Human NER involves the ordered action of factors in dual incision and DNA repair resynthesis steps (4). Any mutation that affects either the enzymatic activity or the ordered assembly of the dual incision complex leads to genetic disorders such as xeroderma pigmentosum, trichothiodystrophy, or Cockayne syndrome (5, 6).In global genome repair, the dual incision is a multistep process that results from the coordinated action of XPC/ HR23B, TFIIH, XPA, RPA, XPG, and XPF/ERCC1, resulting in the removal of the damaged oligonucleotide (4, 7, 8). After being recognized by the XPC/HR23B complex, the damaged DNA structure is targeted by TFIIH, which recruits the other factors upon the addition of ATP (9 -11). The unwound DNA is then incised by the two endonucleases XPG and XPF/ERCC1 on the 3Ј and 5Ј side of the lesion, respectively (12-15), leaving a gap structure that is filled up by the DNA polymerase ⑀ or ␦ and the accompanying factors PCNA, RF-C, RPA, and DNA ligase I (16). Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19). Although the hypothesis of the sequential assembly, which has gained a lot of support from recent biological studies, seems to be more accepted, the order of assembly of the NER factors on the damaged DNA and their contribution to the DNA remodeling to allow the repair are not fully understood (10,20,21). As an example, to further learn abou...

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.