Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycledependent kinases and TFIIF-dependent phosphatases target the CTD. However, the repetitive nature of the CTD prevents determination of phosphorylated sites by conventional biochemistry methods. Fortunately, a panel of monoclonal antibodies is available that distinguishes between phosphorylated isoforms of RNA polymerase II's (RNAP II) largest subunit. Here, we review how successful these tools have been in monitoring RNAP II phosphorylation changes in vivo by immunofluorescence, chromatin immunoprecipitation and immunoblotting experiments.The CTD phosphorylation pattern is precisely modified as RNAP II progresses along the genes and is involved in sequential recruitment of RNA processing factors. One of the most popular anti-phosphoCTD Igs, H5, has been proposed in several studies as a landmark of RNAP II molecules engaged in transcription. Finally, we discuss how global RNAP II phosphorylation changes are affected by the physiological context such as cell stress and embryonic development.Keywords: RNA polymerase II; CTD-phosphorylation; CTD-kinase; CTD-phosphatase; transcription; mRNA processing.The C-terminal domain (CTD) of RNA polymerase II's (RNAP II) largest subunit is essential for transcription [1][2][3], for its function as enhancer [4,5], for organization of transcription foci within the nucleus [6] and for pre-mRNA processing [7,8]. The CTD consists of multiple repeats of a seven amino acid motif [9,10] (Fig. 1). This motif has been conserved during evolution in eukaryotes, but the number of repeats varies depending on the species: 26 in yeast, 45 in flies and 52 in mammals. Five out of seven amino acids in the consensus motif are phosphate acceptors and indeed phosphorylation is a major post-translational modification of the CTD in vivo [11]. Serine O-glycosylation has also been reported as a minor modification with unknown functional significance [12]. In contrast, CTD phosphorylation plays a major role in the transcriptional process [13][14][15][16]. A large variety of kinases have been reported to phosphorylate the CTD of RNAP II in vitro [17,18]. Of particular significance, CDK7, CDK8 and CDK9 are subunits of the TFIIH general transcription factor, of the mediator complex and of the positive transcription elongation factor (P-TEFb), respectively.Interactions with the unphosphorylated CTD are involved in assembly of RNAP II with the mediator complex to form a holoenzyme or with general transcription factors to form a preinititiation complex of transcription. Phosphorylation of the CTD is required to disrupt these interactions at elongation of transcription and to assist the recruitment of pre-mRNA modification enzymes [16,19].Initial studies generally considered CTD phosphorylation as an all or nothing process. Here, we review recent evidence that multiple forms of CTD-phosphorylated RNAP II are present in cells. These forms have been character...

Increasing evidences suggest that nuclear pore complexes (NPCs) control different aspects of nuclear metabolism, including transcription, nuclear organization, and DNA repair. We previously established that the Nup84 complex, a major NPC building block, is part of a genetic network involved in DNA repair. Here, we show that double-strand break (DSB) appearance is linked to a shared function of the Nup84 and the Nup60/Mlp1-2 complexes. Mutants within these complexes exhibit similar genetic interactions and alteration in DNA repair processes as mutants of the SUMO-protease Ulp1. Consistently, these nucleoporins are required for maintenance of proper Ulp1 levels at NPCs and for the establishment of the appropriate sumoylation of several cellular proteins, including the DNA repair factor Yku70. Moreover, restoration of nuclear envelope-associated Ulp1 in nucleoporin mutants reestablishes proper sumoylation patterns and suppresses DSB accumulation and genetic interactions with DNA repair genes. Our results thus provide a molecular mechanism that underlies the connection between NPC and genome stability.

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60⌬ mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39⌬ partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.