The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.     

RNA recognition by the human polyadenylation factor CstF.

Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.     

The Drosophila homologue of the 64 kDa subunit of cleavage stimulation factor interacts with the 77 kDa subunit encoded by the suppressor of forked gene

During mRNA 3′ end formation, cleavage stimulation factor (CstF) binds to a GU-rich sequence downstream from the polyadenylation site and helps to stabilise the binding of cleavage-polyadenylation specificity factor (CPSF) to the upstream polyadenylation sequence (AAUAAA). The 64 kDa subunit of CstF (CstF-64) contains an RNA binding domain and is responsible for the RNA binding activity of CstF. It interacts with CstF-77, which in turn interacts with CPSF. The Drosophila suppressor of forked gene encodes a homologue of CstF-77, and mutations in it affect mRNA 3′ end formation in vivo. A Drosophila homologue for CstF-64 has now been isolated, both through homology with the human protein and through protein–protein interaction in yeast with the suppressor of forked gene product. Alignment of CstF-64 homologues shows that the proteins have a conserved N-terminal 200 amino acids, the first half of which is the RNA binding domain with the second half likely to contain the CstF-77 interaction domain; a central region variable in length and rich in glycine, proline and glutamine residues and containing an unusual degenerate repeat motif; and then a conserved C-terminal 50 amino acids. In Drosophila, the CstF-64 gene has a single 63 bp intron, is transcribed throughout development and probably corresponds to l(3)91Cd.     

Complex protein interactions within the human polyadenylation machinery identify a novel component

Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.     

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.     

Hu proteins regulate polyadenylation by blocking sites containing U-rich sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.     

Hexameric architecture of CstF supported by CstF-50 homodimerization domain structure

The Cleavage stimulation Factor (CstF) complex is composed of three subunits and is essential for pre-mRNA 3'-end processing. CstF recognizes U and G/U-rich cis-acting RNA sequence elements and helps stabilize the Cleavage and Polyadenylation Specificity Factor (CPSF) at the polyadenylation site as required for productive RNA cleavage. Here, we describe the crystal structure of the N-terminal domain of Drosophila CstF-50 subunit. It forms a compact homodimer that exposes two geometrically opposite, identical, and conserved surfaces that may serve as binding platform. Together with previous data on the structure of CstF-77, homodimerization of CstF-50 N-terminal domain supports the model in which the functional state of CstF is a heterohexamer.     

Regulation of poly(A) site use during mouse B-cell development involves a change in the binding of a general polyadenylation factor in a B-cell stage-specific manner.

During the development of mouse B cells there is a regulated shift from the production of membrane to the secretion-specific forms of immunoglobulin (Ig) mRNA, which predominate in the late-stage or plasma B cells. By DNA transfection experiments we have previously shown that there is an increase in polyadenylation efficiency accompanying the shift to secretion-specific forms of Ig mRNA (C. R. Lassman, S. Matis, B. L. Hall, D. L. Toppmeyer, and C. Milcarek, J. Immunol. 148:1251-1260, 1992). When we look in vitro at nuclear extracts prepared from early or memory versus late-stage or plasma B cells, we see cell stage-specific differences in the proteins which are UV cross-linked to the input RNAs. We have characterized one of these proteins as the 64-kDa subunit of the general polyadenylation factor cleavage-stimulatory factor (CstF) by immunoprecipitation of UV-cross-linked material. The amount of 64-kDa protein and its mobility on two-dimensional gels do not vary between the B-cell stages. However, the activity of binding of the protein to both Ig and non-Ig substrates increases four- to eightfold in the late-stage or plasma cell lines relative to the binding seen in the early or memory B-cell lines. Therefore, the binding activity of a constitutive factor required for polyadenylation is altered in a B-cell-specific fashion. The increased binding of the 64-kDa protein may lead to a generalized increase in polyadenylation efficiency in plasma cells versus early or memory B cells which may be responsible for the increased use of the secretory poly(A) site seen in vivo.     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage. Received: 6 June 1997 / Accepted: 5 November 1997     

Locked tether formation by cooperative folding of Rna14p monkeytail and Rna15p hinge domains in the yeast CF IA complex

The removal of the 3' region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.     

Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors

Cleavage stimulation factor (CstF) is a heterotrimeric protein complex essential for polyadenylation of mRNA precursors. The 77 kDa subunit, CstF-77, is known to mediate interactions with the other two subunits of CstF as well as with other components of the polyadenylation machinery. We report here the crystal structure of the HAT (half a TPR) domain of murine CstF-77, as well as its C-terminal subdomain. Structural and biochemical studies show that the HAT domain consists of two subdomains, HAT-N and HAT-C domains, with drastically different orientations of their helical motifs. The structures reveal a highly elongated dimer, spanning 165 A, with the dimerization mediated by the HAT-C domain. Light-scattering studies, yeast two-hybrid assays, and analytical ultracentrifugation measurements confirm this self-association. The mode of dimerization and the relative arrangement of the HAT-N and HAT-C domains are unique to CstF-77. Our data support a role for CstF dimerization in pre-mRNA 3' end processing.     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage.     

A novel complex from bovine visual cells of a 33,000-dalton phosphoprotein with beta- and gamma-transducin: purification and subunit structure

Photoreceptors of mammalian retinas contain a 33-kDa (33K) protein that is phosphorylated, in vitro, by cyclic nucleotide dependent protein kinases. The 33K protein is phosphorylated in the dark, in situ, and dephosphorylated upon illumination. The soluble 33K protein from bovine retinas has been purified to near homogeneity by extraction at pH 5.7 and chromatography on ion-exchange, gel filtration, and hydroxylapatite columns. In the native conformation, the 33K protein is associated with a 37-kDa (37K) and a 10-kDa (10K) protein, forming a trimeric complex with a sedimentation coefficient of 4.9 S and an apparent molecular mass of 77 kDa. The 33K protein can be dissociated from the 37K/10K complex by centrifugation in the presence of high pH and high salt; the subunits reassociate to form the trimeric complex upon recentrifugation in an isotonic buffer with neutral pH. The 33K protein is phosphorylated rapidly by exogenous kinase, in vitro, whereas the 37K and 10K subunits remain unphosphorylated. The 37K and 10K subunits cross-react with antibodies prepared against the beta- and gamma-subunits, respectively, of bovine transducin, indicating that the 37K and 10K subunits are immunologically identical with beta- and gamma-transducin, respectively. No immuno-cross-reactivity was observed between the 33K protein and an antibody against the alpha-subunit of bovine transducin. The 33K-beta-/gamma-transducin complex exhibits striking similarity to transducin in its subunit structure and mode of subunit interaction, suggesting it may play an important role in the metabolism and function of rod photoreceptor cells.     

The structure of the CstF-77 homodimer provides insights into CstF assembly

The cleavage stimulation factor (CstF) is essential for the first step of poly(A) tail formation at the 3' ends of mRNAs. This heterotrimeric complex is built around the 77-kDa protein bridging both CstF-64 and CstF-50 subunits. We have solved the crystal structure of the 77-kDa protein from Encephalitozoon cuniculi at a resolution of 2Å. The structure folds around 11 Half-a-TPR repeats defining two domains. The crystal structure reveals a tight homodimer exposing phylogenetically conserved areas for interaction with protein partners. Mapping experiments identify the C-terminal region of Rna14p, the yeast counterpart of CstF-77, as the docking domain for Rna15p, the yeast CstF-64 homologue.     

Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing

mRNA 3′ processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3′ processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3′ processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another''s expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3′ processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3′ processing factors and provided new insights into the regulatory mechanisms of mRNA 3′ processing.     

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.     

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.