Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.     

Poly(A), poly(A) binding protein and the regulation of mRNA stability

This review has focused on the possibility that interactions between mRNA sequences and the poly(A)-nucleoprotein complex play important roles in mRNA turnover. It is important to stress that additional genetic and biochemical tests are necessary to characterize how PABP interacts with mRNA in cells and to determine whether the poly(A) protection hypothesis is accurate. Moreover, there may be a significant number of mRNAs whose half-lives are independent of polyadenylation. For example, the stabilities of poly(A)-containing and deadenylated alpha 2u-globulin and interferon mRNAs are similar in microinjected oocytes. Thus, an important challenge in this field will be to analyse the complex and interactive factors that determine the half-lives of specific mRNAs.     

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).     

Insulin-like growth factor binding protein 1 (IGFBP-1) levels in Turner syndrome.

We tested whether IGFBP-1, a modulator of IGF-I action, would play a role in the pathogenesis of growth failure and metabolic picture of Turner syndrome. Fasting serum levels of IGFBP-1 were assessed in nineteen girls with Turner syndrome (aging 6.5 to 17.2 years) by radioimmunoassay. Our patients showed normal values of IGFBP-1 (mean +/- SD: 68.6 +/- 32.5 micrograms/l, range: 16 to 134 micrograms/l; range for age and pubertal stage-matched normal children: 15 to 180 micrograms/l). IGFBP-1 levels inversely correlated with bone age (p < 0.05), weight (p < 0.001), percentage of ideal body weight (p < 0.002) and body mass index (BMI) (p < 0.001). Our results seem to rule out a role of IGFBP-1 in the pathogenesis of growth failure in Turner syndrome. The close inverse relationship between IGFBP-1 levels and BMI suggests the serum concentrations of IGFBP-1 to be regulated by the nutritional status. Due to IGFBP-1 inhibiting action on IGF biological activity, the reduction of IGFBP-1 levels in overweight subjects might represent a mechanism to enhance the IGF insulin-like activity, thus supplementing the insulin action.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Polypyrimidine tract binding protein modulates efficiency of polyadenylation

Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3' end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3' end processing.     

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1

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Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.     

Polyalanine-independent conformational conversion of nuclear poly(A)-binding protein 1 (PABPN1)

Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.     

Acyl-coenzyme A binding protein (ACBP)

Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four alpha-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located at the helix-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability.     

Specificity of nuclear protein binding to a CYP1A1 negative regulatory element

Primary cultures of rat epidermal keratinocytes lose the ability to respond to chemicals with the induction of CYP1A1 gene expression after approximately 15 passages. This repression is mediated by a CT-rich direct repeat negative regulatory DNA (NeRD) element present in the upstream regulatory region of the CYP1A1 gene. Competitive gel retardation analysis using keratinocyte nuclear extracts and mutant NeRD oligonucleotides revealed the presence of two specific protein-NeRD complexes and revealed the specific nucleotides important for the formation of each complex. These studies demonstrate that these two factors bind to overlapping sites within the NeRD element. Nucleotide specificity of complex A formation is similar to that of previously identified nuclear silencing factors, while that of complex B appears to represent a unique CT-rich binding factor. These results suggest that repression of CYP1A1 gene expression in high passage keratinocytes may involve the interplay between at least two specific NeRD binding factors.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 5′ splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 5′ splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.