A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II

Abstract

Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93: [6975][6976][6977][6978][6979][6980] 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr 1 Ser 2 Pro 3 Thr 4 Ser 5 Pro 6 Ser 7 . In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) consists of multiple repeats of the heptapeptide sequence YSPTSPS (2, 24). The CTD is found in pol II from a variety of species but is not present on pol I or pol III, indicating a role in mRNA biogenesis (22,23). Deletion studies have demonstrated that the CTD is essential for cell viability in yeast, Drosophila, and mammalian cells (3,4,55,75). While roles in transcription activation, enhancer function, and pre-mRNA processing have been proposed, the essential role of the CTD remains elusive.The CTD contains an uncommonly high density of potential phosphorylation sites (26,27). Both hyper-and hypophosphorylated pol II can be detected in vivo, but the distribution of phosphates among the more than 100 potential sites has not been determined. Substitution of alanine for serines in either position 2 or 5 of each repeat is lethal in yeast, suggesting that phosphorylation of both of these sites is essential (70).In vitro transcription experiments have led to the hypothesis that the CTD is reversibly phosphorylated with each transcription cycle (26,27). One role of phosphorylation may be to disrupt contacts between the unphosphorylated CTD and components of the holoenzyme. In addition, phosphorylation may prepare the CTD for a role subsequent to initiation. Indeed, transcription elongation is CTD dependent (1, 44) and is temporally linked to CTD phosphorylation (1,31,44,56,69).Yeast two-hybrid screens have led to the identification of a family of CTD-binding proteins containing serine/arginine-rich motifs (17,64,74). The SCAFs (SR-like CTD-associated factors) contain conserved N-terminal or C-terminal CTD-interacting domains (25). We have previo...

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomycescerevisiae

“…Nrd1 has been reported to associate with the CTD in a yeast two-hybrid assay (7), and recent genetic evidence supports this finding (27). Likewise, a member of the rat SR (serine-arginine-rich)-like CTD-associated factor family, SCAF8 (previously known as rA8) has been shown to bind to the CTD by both genetic and biochemical experiments (7,28). Whereas the sequence homology shared by Pcf11p with the above two proteins is confined to the CTD-binding domain, extensive similarity with all of the other hypothetical proteins shown in the alignment is exhibited (Fig.…”

“…Neither Clp1p (21) nor Pcf11p displays any similarity to known mammalian processing factors. However, Pcf11p does contain a putative CTD-binding domain at the N terminus that is homologous to domains found in a number of polypeptides from various organisms (26)(27)(28).Previous studies in our laboratory have revealed that subunits of CF IA are directly involved in transcription termination: Birse et al (29) demonstrated that mutations in RNA15, RNA14, and PCF11 affect transcription termination based on transcription run-on analysis. Exactly how these factors affect transcriptional termination has yet to be elucidated.…”

“…Neither Clp1p (21) nor Pcf11p displays any similarity to known mammalian processing factors. However, Pcf11p does contain a putative CTD-binding domain at the N terminus that is homologous to domains found in a number of polypeptides from various organisms (26)(27)(28).…”

Trimethylation of Histone H3 Lysine 4 is an Epigenetic Mark at Regions Escaping Mammalian X Inactivation

“…To determine if there are regions that escape X inactivation during spermatogenesis, we used an antibody for the phosphorylated form of RNA polymerase II at serine 2 which is only present in transcriptionally active chromatin. 17,28,29 We found RNA polymerase II to be excluded from the XY body at pachytene, except for several discrete regions (Fig. 5B).…”

Synergie entre les complexes de transcription et de maturation des ARN messagers

“…Enfin, notons que la protéine SCAF8 semble interagir spécifiquement avec le domaine CTD phosphorylé. Des travaux de localisation démontrent clairement que la protéine se retrouve au site de transcription [26]. Toutes ces données appuient le rôle des protéines SCAF dans l'établissement de liens entre la transcription et l'épissage.…”