The C-terminal domain of RNA polymerase II couples mRNA processing to transcription

Abstract

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

Protein factors in pre-mRNA 3′-end processing

“…Both CstF-50 and CstF-77 bind specifically to the CTD of PolII but CstF-50 binds with a higher efficiency [85]. This binding is significantly reduced upon deletion of the first 91 amino acids of CstF-50, indicating that the WD-40 repeats are not sufficient for interaction.…”

“…It is an essential factor in yeast and mammals that interacts directly with pre-mRNA to direct the cleavage reaction, and it is also required for polyadenylation. Besides its role in 3′-end processing, CPSF-160 associates with factors involved in transcriptional initiation (TFIID) [84] and elongation (PolII CTD) [85], and plays a role in transcriptional termination [4]. CPSF-160 is also involved in cytoplasmic polyadenylation in Xenopus oocytes [32].…”

“…Yeast two-hybrid assays showed that the HAT-C domain mediates interactions with the second β-propeller of CPSF-160, the same region that recognizes the AAUAAA PAS [152]. CstF-77 binds specifically to the CTD of PolII but with less efficiency than CstF-50 [85]. Rna14p binds to unphosphorylated CTD, but the binding increases upon phosphorylation of the CTD [157].…”

“…PolII was identified in 3′-end processing because transiently transfected cells with CTD truncations exhibit inefficient polyadenylation [85]. Purified phosphorylated and unphosphorylated CTD activated a reconstituted cleavage reaction in vitro [212].…”

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

“…CstF2 interacts directly with the mRNA, and cells deficient in CstF2 undergo cell cycle arrest and apoptotic death (Takagaki and Manley, 1998). Both the CstF1 and CstF3 subunits interact specifically with the C-terminal domain of RNA polymerase II, likely facilitating the RNA polymerase II-mediated activation of 3 0 end processing (McCracken et al, 1997;Hirose and Manley, 1998). After DNA damage, mRNA 3 0 processing is inhibited as a result of CstF/BARD1/BRCA1 complex formation (Kleiman and Manley, 1999) and of the proteasome-mediated degradation of RNA polymerase II (Kleiman et al, 2005), suggesting the existence of possibly redundant mechanisms to explain the inhibitory effect of UV irradiation.…”