Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation Jessica L. Dermody1, Jonathan M. Dreyfuss5, Judit Ville´ n3, Babatunde Ogundipe2, Steven P. Gygi3, Peter J. Park5, Alfred S. Ponticelli4, Claire L. Moore2, Stephen Buratowski1, Miriam E. Bucheli1* 1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 3 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York, United States of America, 5 Harvard-Partners Center for Genetics and Genomics, Boston, Massachusetts, United States of America Abstract The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation. Citation: Dermody JL, Dreyfuss JM, Ville´n J, Ogundipe B, Gygi SP, et al. (2008) Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation. PLoS ONE 3(9): e3273. doi:10.1371/journal.pone.0003273 Editor: Caroline M. Kane, University of California, United States of America Received August 12, 2008; Accepted September 1, 2008; Published September 26, 2008 Copyright: 2008 Dermody et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants 1K01CA115515-01A1 to M.B., GM51124 to A.S.P., GM46498 and GM56663 to S.B., GM68887 to C.M., and fellowship 5F31GM075383-03 to J.D. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Miriam_Bucheli@hms.harvard.edu Introduction Gene expression is regulated through a complex series of events that coordinate the synthesis, processing and export, and in some instances, degradation of the mRNA. As the polymerase reaches the end of the gene, these events must coincide to produce a functional mRNA. The transcription of sequences that specify cleavage/polyadenylation signals directs the termination phase of transcription [1,2]. In yeast, the cleavage/polyadenylation ma- chinery, which is composed of the Cleavage Factor I (CFI) (CSTF in humans) and Cleavage/Polyadenylation Factor (CPF) (CPSF in humans) complexes, is responsible for the recognition of polyA/ termination signals and their subsequent processing [2–6]. Within CFI, Rna15, the homolog to mammalian CSTF64, specifically recognizes and binds sequences that direct the polyA/termination machinery to the 39 end [7]. Lacking is a detailed understanding of how the recognition of polyA signals transitions towards transcription termination, and how this is linked to the preceding elongation phase. The allosteric or anti-termination model takes into account some of these events. In this model, association of a positive elongation factor or an anti-terminator with RNA Polymerase II (RNAP II) is essential. Dissociation of this factor from the polymerase at the end of a gene is predicted to have two effects: (1) it destabilizes transcriptionally active RNAP II, and (2) it promotes the recruitment of the polyA/termination machinery. The SR-like protein Npl3 functions in transcription, 39 end processing, hnRNP formation, and mRNA export [8–12]. Previ- ously, we proposed that the competition between Npl3 and Rna15 for binding to RNA targets is central to the function of Npl3 in termination and 39 end processing [11,12]. Increased binding of Npl3 to a target sequence can shift the competition away from processing, strongly inhibiting cleavage/polyadenylation in vitro [12]. In vivo, mutations or deletion of npl3 can result in the utilization of weak or otherwise poorly recognized polyA sites, while strong polyA signals are recognized and stably bound by cleavage/polyadenyla- tion factors for processing [11,13]. As we demonstrated previously, shifting the competition in favor of protection instead of processing by overexpressing Npl3 strongly inhibits cleavage/polyadenylation [12]. These results are consistent with a model of competition driven by the relative binding affinities of Rna15 and Npl3, yet an additional layer of regulation could not be excluded. Phosphorylation plays an important role in the association/ dissociation of a number of activators and factors that regulate transcription and mRNA processing. Most notable is the co- transcriptional phosphorylation of the carboxy terminal domain (CTD) of RNAP II. In S. cerevisiae, phosphorylation of the CTD repeat at serine 5 recruits capping enzyme, while phosphorylation at serine 2 functions to coordinate transcription and mRNA processing [14–17]. Another prominent transcription/RNA pro- cessing kinase is Casein Kinase 2 (CK2). In yeast, CK2 associates with elongation factors Spt16/Pob3 and Chd1 [18,19]. In higher eukaryotes, one third of CK2 phosphorylation targets are involved in gene expression, half of these being transcription factors [20]. These include the activator PC4 (Sub1 in yeast), which is required PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3273