How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes

Essential eukaryal mRNA processing events are targeted to nascent transcripts made by RNA polymerase II (Pol2) via physical interactions of the processing machineries with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol2. The Pol2 CTD consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The inherently plastic Pol2 CTD structure is sculpted by dynamic phosphorylation and dephoshorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis–trans isomerization of the prolines. With up to 128n potential Pol2 CTD primary structures (where n is the number of heptads), the Pol2 CTD provides information about the state of the transcription complex (a CTD code) that is read by diverse Pol2 CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation (Buratowski 2009; Corden 2013; Eick and Geyer 2013; Geronimo et al. 2013). The individual amino acids of the Pol2 CTD heptad and their phosphorylation marks are coding “letters” that have distinct outputs with respect to receptor recognition and impact on cellular gene expression (Schwer et al. 2014). The number of heptad repeats comprising the Pol2 CTD, the minimal number of repeats required for viability, and the prevalence of nonconsensus coding letters tend to increase with progression from unicellular to multicellular eukarya, presumably as a means to orchestrate the actions of increasing numbers of Pol2 CTD receptors. We envision a core Pol2 CTD code that governs events common to most eukarya; for example, the essential function of the Ser5-PO4–Pro6 coding “word” in recruiting mRNA capping enzymes to the Pol2 elongation complex (Schwer and Shuman 2011; Schwer et al. 2012), which is conserved in budding yeast, fission yeast, and mammals, notwithstanding major differences in the genetic and physical organization of the capping apparatus in these taxa (Lima et al. 1999; Changela et al. 2001; Pei et al. 2001; Fabrega et al. 2003; Ghosh et al. 2011). The RNA capping enzymes are the first processing factors to act on the growing Pol2 transcript; indeed, capping can commence after synthesis of only a 19- to 22-mer nascent RNA, the point at which the 5′ triphosphate RNA end is extruded from the RNA-binding pocket of elongating Pol2 and becomes accessible to the capping enzymes (Chiu et al. 2002). The m7GpppN cap is formed by three successive enzymatic reactions: (1) RNA triphosphatase (TPase) hydrolyzes the RNA 5′ triphosphate end (pppRNA) to a diphosphate (ppRNA) plus inorganic phosphate, (2) GTP:RNA guanylyltransferase (GTase) converts ppRNA to GpppRNA via a covalent GTase-(lysyl-Nζ)–GMP intermediate, and (3) AdoMet:RNA(guanine-N7)-methyltransferase (MTase) converts GpppRNA to m7GpppRNA (Gu and Lima 2005). Whereas it had long been assumed that capping is a constitutive and efficient process for most mRNAs, this view may be too simplistic. The existence of factors that can stimulate capping of specific transcripts and the discovery of enzymes that mediate the decay of partially processed RNA intermediates in the cap synthetic pathway suggest that capping efficiency is a dynamic process (Chiu et al. 2002; Jiao et al. 2010, 2013; Chang et al. 2012). One potential way to regulate capping is by influencing the timely recruitment of the capping enzymes to the Pol2 elongation complex. A conserved theme among eukarya is the direct binding of the GTase component to the Ser5-PO4 form of the Pol2 CTD (Ho and Shuman 1999; Fabrega et al. 2003; Ghosh et al. 2011). In mammals and budding yeast, the TPase components are recruited passively to the Pol2 CTD by virtue of their physical association with the GTase: in cis as a covalently fused TPase–GTase enzyme, Mce1, in mammals (Ho and Shuman 1999; Changela et al. 2001) or in trans as separately encoded subunits of a TPase•GTase complex in budding yeast (Ho et al. 1999; Gu et al. 2010). The fission yeast Schizosaccharomyces pombe has a distinctive strategy for targeting cap formation to Pol2 transcripts whereby the TPase (Pct1) and GTase (Pce1) enzymes are not associated physically but instead bind independently to the Ser5-phosphorylated Pol2 CTD (Pei et al. 2001). Mammalian and fungal capping enzymes can also gain access to nascent Pol2 transcripts via physical interactions with the transcription elongation factor Spt5 (Wen and Shatkin 1999; Pei and Shuman 2002; Pei et al. 2006; Lidschreiber et al. 2013). Spt5 is a large polypeptide (∼1000–1200 amino acids) composed of multiple domain modules that associates with the Pol2 transcription complex shortly after initiation and can exert negative and positive effects on transcription elongation (Hartzog and Fu 2013). Fission yeast Spt5 has a distinctive C-terminal repeat domain (the “Spt5 CTD”) composed of 18 repeats of a nonapeptide motif (consensus: T1P2A3W4N5S6G7S8K9) that (1) binds the fission yeast RNA capping enzymes Pct1 and Pce1 (Pei and Shuman 2002) and (2) is targeted for threonine phosphorylation by the fission yeast Cdk9 kinase (Pei and Shuman 2002, 2003; Viladevall et al. 2009). Alignment of Spt5 CTD elements in metazoan and fungal species reveals substantive differences, although each contains a series of Thr–Pro or Ser–Pro dipeptide motifs followed by a hydrophobic side chain two residues downstream (Pei and Shuman 2002). The hydrophobic side chain is typically tryptophan in S. pombe; this position is substituted to a tyrosine or histidine in humans. Available genetic evidence points to overlapping roles of the CTDs of fission yeast Pol2 and Spt5 in recruiting the capping enzymes in vivo (Schneider et al. 2010). Unlike the capping enzyme•Pol2 CTD interactions, which stringently depend on the Ser5-PO4 mark, the binding of fission yeast Pct1 and Pce1 to the Spt5 CTD is independent of Thr1 phosphorylation (Pei et al. 2001; Pei and Shuman 2002). The structural principles underpinning capping enzyme•Pol2 CTD interactions were initially elucidated via cocrystallization of Candida albicans GTase (Cgt1) and mammalian GTase (Mce1) bound to Ser5-phosphorylated Pol2 CTD peptide ligands (Fabrega et al. 2003; Ghosh et al. 2011). These two cellular GTases are structurally homologous enzymes composed of two domains: (1) an N-terminal nucleotidyltransferase (NTase) module that contains the guanylate-binding pocket and (2) a C-terminal OB-fold module. A comparison of the Candida and mammalian GTase•Pol2 CTD structures was most revealing because, although the docking sites for Pol2 CTD lie on the surface of the NTase domain in both cases, the docking sites are physically distinct and have little overlap, the atomic contacts to Pol2 CTD are different, and virtually none of the Pol2 CTD-interacting side chains of mammalian GTase are conserved in the Candida enzyme (Ghosh et al. 2011). Thus, capping enzymes from different taxa have evolved different strategies to read the Pol2 CTD code. Here we extend the structural analysis to the GTase enzyme of fission yeast and its interactions with the CTDs of Pol2 and Spt5. We report crystal structures of Pce1 bound to Pol2 CTD and Spt5 CTD ligands. Key findings are that (1) the CTDs of Pol2 and Spt5 interact with completely distinct sites on the NTase and OB domains of the fission yeast GTase, respectively, and (2) whereas the interface of GTase with the Pol2 CTD is dependent on Ser5 phosphorylation, GTase binding to Spt5 CTD is antagonized by Thr1 phosphorylation. We probed by structure-guided mutagenesis the contributions of the GTase•CTD interfaces to CTD binding in vitro and capping enzyme function in vivo. Our findings cohere into a model in which the capping enzyme recognizes and responds to distinct coding cues and phosphorylation marks in the Pol2 and Spt5 CTDs.