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. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Gene accession numbers associated with this paper: AY140902, AY140900, AY168923, AY140901     

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.     

A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [³²P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.     

Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.     

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     

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

Background  

The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits.     

An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDa subunit of CPSF

The Arabidopsis genome possesses a number of sequences that are predicted to encode proteins that are similar to mammalian and yeast polyadenylation factor subunits. One of these resides on chromosome V and has the potential to encode a polypeptide related to the 100 kDa subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF). This gene encodes a ca. 2400 nucleotide mRNA that in turn can be translated to yield a polypeptide that is 39% identical to the mammalian CPSF100 protein. Antibodies raised against the Arabidopsis protein recognized distinctive polypeptides in nuclear extracts prepared from pea and wheat germ, consistent with the hypothesis that the Arabidopsis protein is resident in a nuclear polyadenylation complex. Interestingly, the Arabidopsis CPSF100 was found to interact with a portion of a nuclear poly(A) polymerase. This interaction was attributable to a 60 amino acid domain in the CPSF100 polypeptide and the N-terminal 220 amino acids of the poly(A) polymerase. An analogous interaction has yet to be described in other eukaryotes. The interaction with PAP thus indicates that the plant CPSF100 polypeptide is likely part of the 3-end processing machinery, but suggests that this complex may function differently in plants than it does in mammals and yeast.     

Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation

Cytoplasmic polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before polyadenylation begins. The identification of these factors has broad implications for biological process that employ polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.     

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.     

Arabidopsis CLP1-SIMILAR PROTEIN3, an ortholog of human polyadenylation factor CLP1, functions in gametophyte, embryo, and postembryonic development

Polyadenylation factor CLP1 is essential for mRNA 3'-end processing in yeast and mammals. The Arabidopsis (Arabidopsis thaliana) CLP1-SIMILAR PROTEIN3 (CLPS3) is an ortholog of human hCLP1. CLPS3 was previously found to be a subunit in the affinity-purified PCFS4-TAP (tandem affinity purification) complex involved in the alternative polyadenylation of FCA and flowering time control in Arabidopsis. In this article, we further explored the components in the affinity-purified CLPS3-TAP complex, from which Arabidopsis cleavage and polyadenylation specificity factor (CPSF) subunits AtCPSF100 and AtCPSF160 were found. This result implies that CLPS3 may bridge CPSF to the PCFS4 complex. Characterization of the CLPS3 mutant revealed that CLPS3 was essential for embryo development and important for female gametophyte transmission. Overexpression of CLPS3-TAP fusion caused a range of postembryonic development abnormalities, including early flowering time, altered phyllotaxy, and abnormal numbers and shapes of flower organs. These phenotypes are associated with the altered gene expression levels of FCA, WUS, and CUC1. The decreased ratio of FCA-beta to FCA-gamma in the overexpression plants suggests that CLPS3 favored the usage of FCA regular poly(A) site over the alternative site. These observations indicate that Arabidopsis CLPS3 might be involved in the processing of pre-mRNAs encoded by a distinct subset of genes that are important in plant development.