The Essential N Terminus of the Pta1 Scaffold Protein Is Required for snoRNA Transcription Termination and Ssu72 Function but Is Dispensable for Pre-mRNA 3′-End Processing

Abstract

Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-⌬75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-⌬75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

“…2) and a Nterminal Symplekin HEAT domain interacting with the RNAPII CTD phosphatase Ssu72, but do not incorporate C-terminal CPSF73, CPSF100, and Symplekin regions important for CCC formation. Additionally, previous bioinformatics, yeast-two hybrid and pull-down analyses have led to hypotheses that the C-termini of CPSF100, CPSF73, and Symplekin interact (Jenny et al 1996;Kyburz et al 2003;Dominski et al 2005b;Zhelkovsky et al 2006;Mandel et al 2008;Ghazy et al 2009), but these studies never included all three CCC components, instead focusing on only two factors. The experiments described here are the first systematic, in vivo approach to defining simultaneous CPSF73, CPSF100, and Symplekin binding interactions in the CCC.…”

“…This N-terminal HEAT domain in human Symplekin mediates proteinprotein interactions and interacts directly with Ssu72, a RNA polymerase II carboxy-terminal domain (RNAPII CTD) Ser 5 phosphatase that is required for mRNA 3 ′ end processing in yeast (He et al 2003;Krishnamurthy et al 2004;Xiang et al 2010). The C-terminal 85 amino acids of the Symplekin yeast homolog, Pta1, interact with Ysh1 (the yeast CPSF73 homolog) in a directed yeast-two hybrid assay and full-length Pta1 interacts with yeast CPSF100 in an in vitro pull-down assay (Ghazy et al 2009). Additionally, full-length Pta1 interacts directly with the C-terminus of Ysh1 (Zhelkovsky et al 2006).…”

A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation

“…with other subunits of the processing complex. Since Fip1 has been reported to interact with Rna14 of CF I and Pta1 of CPF in vitro (Preker et al 1995;Ghazy et al 2009), we tested whether these interactions were dependent on the flexible linker of Fip1. For these assays, we used two C-terminal truncations of Fip1-one which included the linker region (Fip1, 1-206) and one which completely lacked it (Fip1, 1-105)-in pull-downs with either GST-tagged Pta1 or MBP-tagged CF IA, which is a subcomplex of CF I that contains Rna14, Rna15, Clp1, and Pcfll, but lacks the Hrp1 protein (Kessler et al 1996).…”

“…All protein expression was performed in E. coli Rosetta (DE3), and expression and purification was performed as described . GST pull-downs were performed using a modification of the protocol described by Ghazy et al (2009). GST-tagged proteins were incubated with previously washed glutathione-Sepharose beads in 200 mL of buffer IP-150 (10 mM Tris [pH 7.9], 150 mM NaCl, 0.25M EDTA, 10% glycerol, 1M DTT, 1% NP-40, and protease inhibitors) for 2 h at 4°C, with gentle shaking.…”

Transcription termination by nuclear RNA polymerases

“…2; Morlando et al 2002;Carneiro et al 2008). Additionally, mutations in a subcomplex of the cleavage/ polyadenylation machinery, the APT complex (associated with Pta1 [homolog of symplekin]), lead to read-through transcription of certain sn/snoRNA genes (Dheur et al 2003;Ganem et al 2003;Nedea et al 2003;Steinmetz and Brow 2003;Kim et al 2006;Ghazy et al 2009). The APT factors implicated in sn/snoRNA termination include Pti1, a second yeast homolog of CstF-64 that also binds Rna14; Ref2, which interacts with the snoRNPspecific protein Nop1 (Morlando et al 2004); the CTD phosphatase Ssu72 (Ganem et al 2003;Steinmetz and Brow 2003;Kim et al 2006); and Swd2, which is also a component of the Set1 complex (Cheng et al 2004;Dichtl et al 2004).…”