The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation

Abstract

Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3'-end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3'-end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3'-end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3'-end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Protein factors in pre-mRNA 3′-end processing

“…The C-terminus of CPSF-73 does interact with other proteins in the 3′-end processing complex, including CPSF-100 [117] and Pta1p [111]. Brr5p/Ysh1p has also been shown to interact strongly with Clp1p, but the binding region is unknown [118]. This association may bridge CPSF with CF II m in mammals [119].…”

“…Cft2p/Ydh1p is critical for yeast cell viability [121], and conditional mutants of this protein disrupt both cleavage and polyadenylation in vitro and in vivo [118]. CPSF-100 has recognizable sequence homology to CPSF-73 (23% identity and 49% similarity for their metallo-β-lactamase domains) [103].…”

“…Cft2p/Ydh1p self-associates and also interacts with many proteins of the CPF (Cft1p/Yhh1p, Brr5p/Ysh1p, Pta1p, Pfs2p, Ssu72p, YDL094c), Pcf11p of CF IA, and the CTD of PolII [118]. Deletion mutagenesis studies showed that the C-terminal region of Cft2p/Ydh1p is required for its self-association, while the N-terminal region is required for interaction with Pfs2p.…”

“…Deletion mutagenesis studies showed that the C-terminal region of Cft2p/Ydh1p is required for its self-association, while the N-terminal region is required for interaction with Pfs2p. In contrast, the full-length protein is required for interactions with the other proteins [118]. Yeast two-hybrid studies showed that the last 245 residues of CPSF-73 can interact with CPSF-100 [117].…”

“…This CstF binding region is followed by the Clp1 interacting segment (477-499, discussed below), which is flanked by a zinc finger on each side (N-terminal C 2 H 2 and C-terminal C 2 HC type). Pcf11p also interacts with Cft1p/ Yhh1p, Cft2p/Ydh1p, Brr5p/Ysh1p, and Pta1p [118], but the location of the binding sites is unknown.…”