The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Abstract

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTDbinding proteins may act to physically and functionally link transcription and pre-mRNA processing.The C-terminal domain of the largest subunit of RNA polymerase II (CTD) consists of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (1, 2). Deletion studies demonstrated that the ClD is essential for cell growth (3-6), but the nature of this essential function is not known. The CID is only found on RNA polymerase II (pol II), suggesting that it plays a unique role in mRNA biogenesis (7 Despite identification of interaction partners, the role of the CTD in transcription remains unclear. The CTD is not required for either basalV(14, 15) or activated (16, 17) transcription of some genes in vitro. Furthermore, inhibition of CTD kinase does not block in vitro transcription from the adenovirus major late promoter or from a GAL4 VP16-activated promoter (18,19). Thus, these results indicate that the CTD is not essential for specific initiation at some promoters.CTD function may be required for postinitiation steps in the biogenesis of mRNA. O'Brien et al. (20) have demonstrated that several genes contain paused pol IIA complexes that can reenter the elongation mode coincident with CTD phosphorylation. In yeast, CTD-truncated pol II synthesizes an excess of GAL4-induced promoter proximal transcripts (D. L. Bentley, personal communication). Thus, these results argue that the CTD plays an important role subsequent to initiation. While the CTD has previously been proposed to function in premRNA processing (refs. 7 and 21 and H. Rienhoff and J. Boeke, personal communication), no experimental data have yet supported these models.We used the yeast two-hybrid system (22) to identify proteins that interact with the CTD. This unbiased approach did not yield proteins that are expected to be involved in transcription initiation, like TBP or the SRBs, but rather a set of proteins similar to RNA processing factors. In this paper we report the identification and characte...

Transcription termination by nuclear RNA polymerases

“…Nrd1 contains a CID (Meinhart and Cramer 2004;Meinhart et al 2005) that interacts with CTD phosphorylated at Ser5 (Conrad et al 2000;Gudipati et al 2008;Vasiljeva et al 2008a). Nrd1 also interacts with another essential RNA-binding protein, Nab3 (Yuryev et al 1996). Nrd1, Nab3, and Sen1 form the Nrd1 complex, which is detected by ChIP at snoRNAs genes (Kim et al 2006) as well as some mRNAs genes, mainly at promoter regions (Nedea et al 2003).…”

“…ChIP analysis showed RNAPII read-through at most snoRNAs genes in a SEN1 mutant strain or a strain defective for its helicase activity (Kim et al 2006;Steinmetz et al 2006b). Sen1 associates with the CTD and also coprecipitates with several snoRNAs and some snRNAs (Yuryev et al 1996;Ursic et al 1997Ursic et al , 2004. Nedea et al (2008) showed that Glc7, the yeast PP1 (protein phophatase-1) catalytic subunit, is also a regulator of snoRNA termination, interacting directly with Sen1.…”

Exogenous cdk4 overcomes reducedcdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF – a model for overcoming inhibition of proliferation by CBF oncoproteins

“…Band`a' represents Hsp73, which may signify an artifact of our system as the ER domain interacts with the very closely related Hsp70 protein (Smith and Toft, 1993). Band`b' includes a clone for an RNA polymerase II-binding protein (Yuryev et al, 1996) and a clone for subunit of RNA polymerase III (Sepehri and Hernandez, 1997), both of which potentially regulate cell proliferation. Another clone isolated from band`b' encodes fau, an arsenateresistance protein which is also a tumor suppressor (Rossman and Wang, 1999).…”

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

“…These early genomic efforts suggested that, similar to DNA binding proteins, functional groupings of genes are being bound by RNA binding proteins perhaps as post-transcriptional operons (Keene and Tenenbaum 2002). However, it is not known at which stage of RNA processing these operons are established.Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al 1996;Yuryev et al 1996;Kim et al 1997). The CTD is phosphorylated as transcription begins.…”