The Splicing Factor, Prp40, Binds the Phosphorylated Carboxyl-terminal Domain of RNA Polymerase II

Abstract

We showed previously that the WW domain of the prolyl isomerase, Ess1, can bind the phosphorylated carboxyl-terminal domain (phospho-CTD) of the largest subunit of RNA Polymerase II. Analysis of phospho-CTD binding by four other WW domain-containing Saccharomyces cerevisiae proteins indicates the splicing factor, Prp40, and the RNA polymerase II ubiquitin ligase, Rsp5, can also bind the phospho-CTD. The identification of Prp40 as a phospho-CTD binding protein represents the first demonstration of direct interaction between a documented splicing factor and the phospho-CTD. Domain dissection studies reveal that phospho-CTD binding occurs at multiple locations in Prp40, including sites in both the WW and FF domain regions. Because the conserved repeats of the CTD make it an ideal ligand for multi-site binding events, the implications of multi-site binding are discussed. Our data suggest a mechanism by which the phospho-CTD of elongating RNA polymerase II facilitates commitment complex formation by juxtaposing the 5 and 3 splice sites.The carboxyl-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II (1) plays a central role in mRNA synthesis. The CTD is composed of 26 -52 heptad repeats with the consensus sequence, YSPTSPS. These repeats are extensively phosphorylated in RNA polymerase II actively engaged in transcript elongation (2-5).In the past several years evidence has accumulated indicating that the phosphorylated form of the CTD acts to coordinate pre-mRNA processing events (for review see Refs. 6 -10). The 5Ј capping complex is not only localized near the pre-mRNA by direct association with the phospho-CTD (11, 12) but is also allosterically activated by phospho-CTD binding (13,14). A variety of evidence strongly suggests that the phospho-CTD is involved in splicing. The hyperphosphorylated form of RNA polymerase II colocalizes with splicing factors when transcriptionally active (15, 16) and in the nuclear matrix (17-19). Several phospho-CTD binding proteins have been identified that contain SR and RRM domains as found in many splicing factors (20,21). Fusion proteins with a hyperphosphorylated CTD can inhibit splicing in vivo (22). In addition, transcriptionally unengaged but phosphorylated RNA polymerase II is able to stimulate splicing in vitro (23).Recently, we demonstrated that the prolyl isomerase, Ess1, can bind the phosphorylated form of the CTD and that this binding is mediated by its WW domain (24). Because the prolyl isomerase activity of Ess1 preferentially acts on phospho-SerPro peptide bonds, as are found in abundance in the phospho-CTD, our findings provided a plausible explanation for earlier results implicating Ess1 in pre-mRNA 3Ј end formation (25,26).WW domains, named for two highly conserved tryptophan residues, are small independently folding protein domains consisting of slightly more than 30 amino acids arranged in three anti-parallel ␤ sheets (27, 28). These domains have been shown to bind proline-rich sequences containing several different motifs (29). Of five protei...

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…Ssd1 has also been shown to be RNA-associated in vivo (49). One possibility is that its association with the phosphoCTD of elongating Pol II targets Ssd1 to its cognate RNAs in the cell, much as the association with the PCTD is thought to target the splicing factor Prp40 (34). It is also conceivable that Ssd1 performs additional functions in the cell independent of its phosphoCTD association.…”

The RNA polymerase C-terminal domain

“…The S. cerevisiae protein Prp40, which is associated with U1 snRNP, binds to phosphorylated CTD repeats through its WW domain. 29 Phospho-CTD binding appears to be a frequent function of WW domains, and also of the related FF domain. These domains are found on multiple splicing factors and, in addition to interactions with the CTD, also mediate interactions with other splicing factors.…”

RNA Polymerase II Phosphorylation and Gene Expression Regulation

“…Hyperphosphorylated, but not hypophosphorylated RNAPII, has been found associated with splicing factors and detected in active spliceosomes [246][247][248]. For instance, in yeast, the splicing factor Prp40 binds to phosphorylated CTD [249]; in mammals, Spt6 binds selectively to the CTD-Ser2P [112], and the spliceosome-associated protein CA150 interacts with phosphorylated CTD while interacting with the SF1 splicing factor [250][251]. Therefore, all these studies led to the idea that the phosphorylated CTD acts as a scaffold, binding multiple splicing factors, and directly enhancing the spliceosome assembly.…”