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.

The synthesis of mature mRNA in eukaryotes and its utilization in the cytoplasm require cotranscriptional modifications of the pre-mRNA by capping at the 5′ end, removal of introns by splicing, and cleavage at the 3′ end followed by the addition of a poly(A) tail (4, 27, 32). mRNA 3′-end formation is an essential step in mRNA biogenesis and acts at many levels to influence gene expression. Its execution prevents readthrough transcription from interfering with DNA elements such as promoters, centromeres, and replication origins. Without a poly(A) tail, mRNA is targeted for degradation by nuclear surveillance mechanisms, is exported inefficiently from the nucleus, and is poorly translated in the cytoplasm. The maturation of mRNA 3′ ends also serves as an important point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Furthermore, 3′-end processing has been linked to other essential processes such as chromosome segregation, DNA repair, and tissue-specific protein expression (23, 26, 46). mRNA 3′-end formation in Saccharomyces cerevisiae requires the concerted action of two multisubunit factors, cleavage factor (CF) I and cleavage/polyadenylation factor (CPF), that recognize processing signals around the poly(A) site. These complexes are phylogenetically conserved and are comparable to mammalian CstF and CPSF, respectively (27). Cleavage requires CF I and CPF, while tail synthesis requires these factors plus the Pab1 or Nab2 poly(A) binding protein (17). CF I is composed of Rna14, Rna15, Pcf11, Clp1, and Hrp1/Nab4 (21, 22, 35). The holo-CPF complex can be separated into core CPF and the APT subcomplex (29). Core CPF includes Pta1, Cft1, Cft2, Mpe1, Pfs2, Fip1, Pap1, the poly(A) polymerase, and Ysh1/Brr5, the putative pre-mRNA endonuclease (10, 12, 15, 28, 29, 36, 45). The APT subcomplex of CPF includes Pta1, Pti1, Ref2, Swd2, Syc1, and the two phosphatases Ssu72 and Glc7 (29). Even though holo-CPF is important for optimal processing, traditional multistep chromatographic fractionation showed that a smaller complex, called CF II, was sufficient for cleavage in combination with CF I and contained only the Cft1, Cft2, Ysh1, and Pta1 subunits (48). The essential Pta1 subunit was initially defined by a conditional growth mutation, pta1-1, that causes the accumulation of unspliced pre-tRNA in vivo (31). Pta1 is important for both cleavage and poly(A) addition (35, 48), and its phosphorylation inhibits the poly(A) addition step (16). Pta1 also has roles in redirecting the machinery to the 3′ end of nonadenylated snoRNA transcripts (29), in regulating the phosphorylated state of the RNA polymerase II (RNAP II) C-terminal domain (CTD) (25), and in the gene looping that juxtaposes the 5′ and 3′ ends of genes (1). Symplekin, the Pta1 homolog in higher eukaryotes, has been proposed to be a scaffold for assembling the mammalian 3′-end-processing complex (43). Symplekin has also been implicated in the formation of the cleaved, unadenylated ends of replication-dependent histone mRNAs (24), in the cytoplasmic polyadenylation of stored maternal mRNAs in preparation for their translation (2), and in the splicing of tRNA precursors (34). Consistent with a scaffold function, the 90-kDa Pta1 interacts physically and/or genetically with the CPF subunits Ysh1 (the candidate nuclease), Pti1 (thought to suppress CPF's polyadenylation activity on snoRNA transcripts), Ssu72 (an RNAP II CTD serine-5 phosphatase and the only factor dedicated to cleavage), Glc7 (a phosphatase needed for the polyadenylation step), and Syc1 (a negative regulator of mRNA 3′-end formation) (9, 15, 16, 29, 49). However, it is unknown whether all of these contacts are made simultaneously; perhaps some occur sequentially during mRNA synthesis or only in certain types of complexes, while others may never happen inside the cell. The range of Pta1 interactions, especially with three of the enzymes of the complex (Ssu72, Ysh1, and Glc7) and functionally with the fourth, Pap1, suggests that Pta1 occupies a central position within the 3′-end processing complex and helps coordinate its various activities. However, little is known about how Pta1 performs this critical function. In this study, we explore how Pta1 might act as a scaffold protein and show that it uses different regions to contact subunits that are essential to the function of CPF in mRNA 3′-end processing, snoRNA termination, CTD Ser5-P dephosphorylation, and gene looping. In addition, we identify new interactions between Pta1 and CF I, indicating that the organizational role of Pta1 includes making cross-factor connections. We also make the surprising observation that a Pta1 derivative that lacks the essential 300 amino acids at the N terminus and is incapable of interacting with and stabilizing Ssu72 is completely functional in cleavage and polyadenylation. These findings support a model in which the primary function of Ssu72 in mRNA 3′-end processing is to block an inhibitory activity of the Pta1 N-terminal domain.