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At least 19 recordsLinked to original sources
PubMed1992

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.

Animals↗
PubMed1995

The binding of a subunit of the general polyadenylation factor cleavage-polyadenylation specificity factor (CPSF) to polyadenylation sites changes during B cell development.

During the development of mouse B cells there is a regulated shift from the production of membrane (mb) to secretory-specific (sec) forms of immunoglobulin (Ig) mRNA. The mRNAs are produced from one gene that is alternatively processed at the 3' end. We have previously shown that there is an increase in polyadenylation efficiency accompanying the developmentally regulated shift to secretory-specific forms of Ig mRNA by DNA transfection experiments (1). When we look in vitro at nuclear extracts prepared from early/memory versus late stage/plasma B cells, we see cell stage-specific differences in the proteins which are crosslinked to poly(A) site-containing RNAs. Here we show that one of these proteins is the mouse homologue of 100 kDa subunit of Hela CPSF by immunoprecipitation and Western analysis of UV crosslinked material. The amount of 100 kDa protein and its mobility on two-dimensional gels do not change between the B cell stages. However, the binding of the 100 kDa polypeptide to poly(A) sites increases in the late stage/plasma cell lines relative to the binding seen in early/memory cell lines. The increased binding may reflect an increase in polyadenylation efficiency at the sec poly(A) site in plasma cells versus early/memory cells seen in vivo.

Animals↗
PubMed1995

The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation.

Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs. CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis. Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product. Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF. Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure. We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymerase (PAP). These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail. Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays. We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.

Amino Acid Sequence↗
PubMed1997

Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA.

Cleavage of pre-mRNA during 3'-end formation in yeast requires two protein factors, cleavage factor I (CF I) and cleavage factor (CF II). A 5300-fold purification of CF II indicates that four polypeptides of 150, 105, 100, and 90 kDa copurify with CF II activity. The 150-kDa protein is recognized by antibodies against Cft1, the yeast homologue of the 160-kDa subunit of the mammalian cleavage/polyadenylation specificity factor (CPSF). The 100-kDa subunit is identical to Brr5/Ysh1, a yeast protein with striking similarity to the 73-kDa subunit of CPSF. The 105-kDa protein, designated Cft2 (cleavage factor two) exhibits significant homology to the CPSF 100-kDa subunit. Cft2 is cross-linked to pre-mRNA substrate containing the poly(A) site and wild type upstream and downstream flanking sequences, but not to precleaved RNA lacking downstream sequences or to substrate in which the (UA)6 processing signal has been deleted. The specific binding of Cft2 to the RNA substrate is ATP-dependent, in agreement with the requirement of ATP for cleavage. The sequence-specific binding of Cft2 and the similarities of CF II subunits to those of CPSF supports the hypothesis that CF II functions in the cleavage of yeast mRNA 3'-ends in a manner analagous to that of CPSF in the mammalian system. These results provide additional evidence that certain features of the molecular mechanism of mRNA 3'-end formation are conserved between yeast and mammals, but also highlight unexpected differences.

Adenosine Triphosphate↗
PubMed1996

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.

Glutathione Transferase↗
PubMed1997

Creatine phosphate, not ATP, is required for 3' end cleavage of mammalian pre-mRNA in vitro.

The poly(A) tail of a mammalian mRNA is generated by endonucleolytic cleavage and poly(A) addition. Previous studies conducted with nuclear extracts suggested an ATP requirement for the cleavage step. We have reexamined the cofactor requirement, initially with the SV40 late pre-mRNA, which requires for cleavage four protein factors, cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Using highly purified preparations of these factors, which lacked detectable creatine phosphokinase and ATPase activities, creatine phosphate (CP) was, surprisingly, found to be sufficient to promote efficient cleavage. Although other phosphate compounds substituted poorly or not at all for CP, another phosphoguanidine, arginine phosphate, was fully functional. Notably, ATP was neither necessary nor sufficient, and could in fact inhibit the reaction. Treatment of the purified factors with hexokinase plus glucose (to deplete any contaminating ATP) was without effect, as was addition of EDTA. Using 32P-labeled CP, we found that neither hydrolysis of CP nor phosphate transfer from CP occurred during the cleavage reaction. CP also allowed cleavage of the adenovirus 2 L3 pre-mRNA. However, in this case, ATP both enhanced the reaction and influenced the precise site of cleavage, perhaps reflecting the requirement of poly(A) polymerase for cleavage of this RNA. These results indicate that ATP is not essential for 3' pre-mRNA cleavage and that CP or a related compound can function as a necessary cofactor.

Adenosine Triphosphate↗
PubMed1995

Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor.

3'-processing of mRNA precursors depends on several protein factors. One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction. We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal. The cDNAs code for an open reading frame of 1444 amino acids. The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2. Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF. The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs. It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb).

Amino Acid Sequence↗
PubMed2006

The 73 kD subunit of the cleavage and polyadenylation specificity factor (CPSF) complex affects reproductive development in Arabidopsis.

The cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3'-end processing apparatus in eukaryotes. The Arabidopsis genome contains five genes encoding CPSF homologues (AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30). These CPSF homologues interact with each other in a way that is analogous to the mammalian CPSF complex or their yeast counterparts, and also interact with the Arabidopsis poly(A) polymerase (PAP). There are two CPSF73 like proteins (AtCPSF73-I and AtCPSF73-II) that share homology with the 73 kD subunit of the mammalian CPSF complex. AtCPSF73-I appears to correspond to the functionally characterized mammalian CPSF73 and its yeast counterpart. AtCPSF73-II was identified as a novel protein with uncharacterized protein homologues in other multicellular organisms, but not in yeast. Both of the AtCPSF73 proteins are targeted in the nucleus and were found to interact with AtCPSF100. They are also essential since knockout or knockdown mutants are lethal. In addition, the expression level of AtCPSF73-I is critical for Arabidopsis development because overexpression of AtCPSF73-I is lethal. Interestingly, transgenic plants carrying an additional copy of the AtCPSF73-I gene, that is, the full-length cDNA under the control of its native promoter, appeared normal but were male sterile due to delayed anther dehiscence. In contrast, we previously demonstrated that a mutation in the AtCPSF73-II gene was detrimental to the genetic transmission of female gametes. Thus, two 73 kD subunits of the AtCPSF complex appear to have special functions during flower development. The important roles of mRNA 3'-end processing machinery in modulating plant development are discussed.

Arabidopsis↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed2001

RNA polymerase II-dependent positional effects on mRNA 3' end processing in the adenovirus major late transcription unit.

During the early phase of adenovirus infection, the promoter-proximal L1 poly(A) site in the major late transcription unit is used preferentially despite the fact that the distal L3 poly(A) site is stronger (i.e. it competes better for processing factors and is cleaved at a faster rate, in vitro). Previous work had established that this was due at least in part to the stable binding of the processing factor, cleavage and polyadenylation specificity factor, to the L1 poly(A) site as mediated by specific regulatory sequences. It is now demonstrated that in addition, the L1 poly(A) site has a positional advantage because of its 5' location in the transcription unit. We also show that preferential processing of a particular poly(A) site in a complex transcription unit is dependent on RNA polymerase II. Our results are consistent with recent reports demonstrating that the processing factors cleavage and polyadenylation specificity factor and cleavage stimulatory factor are associated with the RNA polymerase II holoenzyme; thus, processing at a weak poly(A) site like L1 can be enhanced by virtue of its being the first site to be transcribed.

Adenoviridae↗
PubMed1997

The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins.

Cleavage and polyadenylation specificity factor (CPSF), a key component of the mammalian RNA 3'-end processing machinery, consists of four subunits of 160, 100, 73, and 30 kD. Here we report the isolation and characterization of a cDNA encoding the 30-kD polypeptide. Antibodies raised against this protein inhibit cleavage and polyadenylation and coimmunoprecipitate the other CPSF subunits. The protein sequence contains five C3H-zinc-finger repeats and a putative RNA-binding zinc knuckle motif at the carboxyl terminus. Consistent with this observation, the in vitro translated 30-kD protein binds RNA polymers with a distinct preference for poly(U). In addition, an essential S. cerevisiae gene, YTH1, was cloned which is 40% identical to CPSF 30K at the protein level. Extracts prepared from a conditional yth1 mutant have normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Efficient polyadenylation activity can be restored by the addition of purified polyadenylation factor I (PF I). We demonstrate that Yth1p is a component of PF I that interacts in vivo and in vitro with Fip1p, a known PF I subunit.

Amino Acid Sequence↗
PubMed1994

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit.

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

Amino Acid Sequence↗
PubMed1994

A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein.

Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.

Amino Acid Sequence↗
PubMed1996

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.

Adenoviridae↗
PubMed1999

The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation.

During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

Amino Acid Sequence↗
PubMed1996

Essential yeast protein with unexpected similarity to subunits of mammalian cleavage and polyadenylation specificity factor (CPSF).

The 3' ends of most eukaryotic messenger RNAs are generated by internal cleavage and polyadenylation. In mammals, there is a strict dependence of both reactions on the sequence AAUAAA, which occurs upstream of polyadenylation [poly(A)] sites and which is recognized by CPSF. In contrast, cis-acting signals for yeast 3'-end generation are highly divergent from those of mammals, suggesting that trans-acting factors other than poly(A) polymerase would not be conserved. The essential yeast protein Brr5/Ysh1 shows sequence similarity to subunits of mammalian CPSF and is required for 3'-end processing in vivo and in vitro. These results demonstrate a structural and functional conservation of the yeast and mammalian 3'-end processing machineries despite a lack of conservation of the cis sequences.

Amino Acid Sequence↗
PubMed2002

Cleavage and polyadenylation specificity factor (CPSF)-derived peptides can induce HLA-A2-restricted and tumor-specific CTLs in the majority of gastrointestinal cancer patients.

To identify CTL-directed antigens in gastrointestinal cancer, we have investigated antigens recognized by the HLA-A2-restricted CTL line established from T cells infiltrating into colon cancer and report herein cleavage and polyadenylation specificity factor (CPSF) as a potent antigen holding peptides capable of inducing CTLs. Five peptides at amino acid positions 250-258, 392-400, 534-542, 1296-1304 and 1359-1368 of CPSF, which were recognized by the CTL line, were found to have the ability to induce HLA-A2-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of the majority (69%, 11/16) of gastrointestinal cancer patients with different HLA-A2 subtypes. Thus, these peptides might be appropriate molecules for use in the peptide-based specific immunotherapy of HLA-A2(+) patients with gastrointestinal cancers.

Amino Acid Sequence↗