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

Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.     

A multisubunit 3'' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.     

Fip1 regulates the activity of Poly(A) polymerase through multiple interactions

Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.     

An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis Fip1 homolog is characterized. The gene for this protein resides on chromosome V and encodes a 1196-amino acid polypeptide. Yeast two-hybrid and in vitro assays indicate that the N-terminal 137 amino acids of the Arabidopsis Fip1 protein interact with poly(A) polymerase (PAP). This domain also stimulates the activity of the PAP. Interestingly, this part of the Arabidopsis Fip1 interacts with Arabidopsis homologs of CstF77, CPSF30, CFIm-25, and PabN1. The interactions with CstF77, CPSF30, and CFIm-25 are reminiscent in various respects of similar interactions seen in yeast and mammals, although the part of the Arabidopsis Fip1 protein that participates in these interactions has no apparent counterpart in other eukaryotic Fip1 proteins. Interactions between Fip1 and PabN1 have not been reported in other systems; this may represent plant-specific associations. The C-terminal 789 amino acids of the Arabidopsis Fip1 protein were found to contain an RNA-binding domain; this domain correlated with an intact arginine-rich region and had a marked preference for poly(G) among the four homopolymers studied. These results indicate that the Arabidopsis Fip1, like its human counterpart, is an RNA-binding protein. Moreover, they provide conceptual links between PAP and several other Arabidopsis polyadenylation factor subunit homologs.     

Novel interactions at the essential N-terminus of poly(A) polymerase that could regulate poly(A) addition in Saccharomyces cerevisiae

Addition of poly(A) to the 3' ends of cleaved pre-mRNA is essential for mRNA maturation and is catalyzed by Pap1 in yeast. We have previously shown that a non-viable Pap1 mutant lacking the first 18 amino acids is fully active for polyadenylation of oligoA, but defective for pre-mRNA polyadenylation, suggesting that interactions at the N-terminus are important for enzyme function in the processing complex. We have now identified proteins that interact specifically with this region. Cft1 and Pta1 are subunits of the cleavage/polyadenylation factor, in which Pap1 resides, and Nab6 and Sub1 are nucleic-acid binding proteins with known links to 3' end processing. Our results suggest a novel mechanism for controlling Pap1 activity, and possible models invoking these newly-discovered interactions are discussed.     

The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.     

Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor

CF II, a factor required for cleavage of the 3' ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides. The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition. In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing. Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex. Extracts prepared from pta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1 substrates and exhibit little processing activity even after prolonged incubation. However, activity is efficiently rescued by the addition of purified CF II to the defective extracts. Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not. The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated. Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I. While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo. The involvement of Pta1 in both steps of mRNA 3'-end formation supports the conclusion that CF II is the functional homolog of CPSF.     

Posttranslational phosphorylation and ubiquitination of the Saccharomyces cerevisiae Poly(A) polymerase at the S/G(2) stage of the cell cycle

The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from alpha-factor-induced G(1) arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G(2) and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.     

The interaction between two Arabidopsis polyadenylation factor subunits involves an evolutionarily-conserved motif and has implications for the assembly and function of the polyadenylation complex

The polyadenylation factor subunit "Factor Interacting with Poly(A) polymerase" (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.     

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.     

Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae

To identify genes involved in poly(A) metabolism, we screened the yeast gene deletion collection for growth defects in the presence of cordycepin (3′-deoxyadenosine), a precursor to the RNA chain terminating ATP analog cordycepin triphosphate. Δpho80 and Δpho85 strains, which have a constitutively active phosphate-response pathway, were identified as cordycepin hypersensitive. We show that inorganic polyphosphate (poly P) accumulated in these strains and that poly P is a potent inhibitor of poly(A) polymerase activity in vitro. Binding analyses of poly P and yeast Pap1p revealed an interaction with a k_D in the low nanomolar range. Poly P also bound mammalian poly(A) polymerase, however, with a 10-fold higher k_D compared to yeast Pap1p. Genetic tests with double mutants of Δpho80 and other genes involved in phosphate homeostasis and poly P accumulation suggest that poly P contributed to cordycepin hypersensitivity. Synergistic inhibition of mRNA synthesis through poly P-mediated inhibition of Pap1p and through cordycepin-mediated RNA chain termination may thus account for hypersensitive growth of Δpho80 and Δpho85 strains in the presence of the chain terminator. Consistent with this, a mutation in the 3′-end formation component rna14 was synthetic lethal in combination with Δpho80. Based on these observations, we suggest that binding of poly P to poly(A) polymerase negatively regulates its activity.     

Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor

Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers. Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3′ end processing. In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro. We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation. Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro. In vitro assays showed that the N-terminus is necessary for both processing steps. Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA–Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1. Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.     

Efficient mRNA Polyadenylation Requires a Ubiquitin-Like Domain,a Zinc Knuckle,and a RING Finger Domain,All Contained in the Mpe1 Protein

Almost all eukaryotic mRNAs must be polyadenylated at their 3′ ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3′-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3′-end processing.     

Trypanosome MTR4 is involved in rRNA processing

The yeast putative RNA helicase Mtr4p is implicated in exosome-mediated RNA quality control in the nucleus, interacts with the exosome, and is found in the ‘TRAMP’ complex with a yeast nuclear poly(A) polymerase (Trf4p/Pap2p or Trf5p) and a putative RNA-binding protein, Air1p or Air2p. Depletion of the Trypanosoma brucei MTR4-like protein TbMTR4 caused growth arrest and defects in 5.8S rRNA processing similar to those seen after depletion of the exosome. TbNPAPL, a nuclear protein which is a putative homolog of Trf4p/Pap2p, was required for normal cell growth. Depletion of MTR4 resulted in the accumulation of polyadenylated rRNA precursors, while depletion of TbNPAPL had little effect. These results suggest that polyadenylation-dependent nuclear rRNA quality control is conserved in eukaryotic evolution. In contrast, there was no evidence for a trypanosome TRAMP complex since no stable interactions between TbMTR4 and the exosome, TbNPAPL or RNA-binding proteins were detected.     

Distinct roles of two Yth1p domains in 3'-end cleavage and polyadenylation of yeast pre-mRNAs

Yth1p is the yeast homologue of the 30 kDa subunit of mammalian cleavage and polyadenylation specificity factor (CPSF). The protein is part of the cleavage and polyadenylation factor CPF, which includes cleavage factor II (CF II) and polyadenylation factor I (PF I), and is required for both steps in pre-mRNA 3'-end processing. Yth1p is an RNA-binding protein that was previously shown to be essential for polyadenylation. Here, we demonstrate that Yth1p is also required for the cleavage reaction and that two protein domains have distinct roles in 3'-end processing. The C-terminal part is required in polyadenylation to tether Fip1p and poly(A) polymerase to the rest of CPF. A single point mutation in the highly conserved second zinc finger impairs both cleavage and polyadenylation, and affects the ability of Yth1p to interact with the pre-mRNA and other CPF subunits. Finally, we find that Yth1p binds to CYC1 pre-mRNA in the vicinity of the cleavage site. Our results indicate that Yth1p is important for the integrity of CPF and participates in the recognition of the cleavage site.