Tristetraprolin Inhibits Poly(A)-Tail Synthesis in Nuclear mRNA that Contains AU-Rich Elements by Interacting with Poly(A)-Binding Protein Nuclear 1

Background

Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol.

Methodology/Principal Findings

To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3''-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element–containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain–deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element–containing tumor necrosis factor α/luciferase mRNA construct.

Conclusion/Significance

In addition to its known cytosolic mRNA–degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element–containing mRNA.     

Poly(A) Tail Length Is Controlled by the Nuclear Poly(A)-binding Protein Regulating the Interaction between Poly(A) Polymerase and the Cleavage and Polyadenylation Specificity Factor

Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, ∼250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of ∼250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of ∼250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.The poly(A) tails present at the 3′ end of almost all eukaryotic mRNAs have two major functions. The first function is in the control of mRNA decay; degradation of the poly(A) tail by a 3′ exonuclease (deadenylation) is the first step in both of the two main pathways of mRNA decay, and the completion of deadenylation triggers the second step, either cap hydrolysis or further 3′–5′ degradation. Because the rate of deadenylation is governed by sequence elements in the mRNA, it is specific for each mRNA species and serves as a major determinant of mRNA half-life (1–3). Obviously, a control of mRNA stability by the rate of deadenylation requires a defined poly(A) length as a starting point. The second function of the poly(A) tail is in the initiation of translation; the cytoplasmic poly(A)-binding protein associated with the poly(A) tail promotes the initiation of translation by an interaction with the initiation factor eIF4G and probably through additional mechanisms (4–7). In this process, poly(A) tail length can also be important. For example, gene regulation during oocyte maturation and early embryonic development of animals depends on translational regulation of maternal mRNAs, and changes in poly(A) tail lengths of specific mRNAs, determined both by deadenylation and by regulated cytoplasmic poly(A) extension, play a major role in this translational regulation. Long poly(A) tails favor translation, whereas a shortening of the tail promotes translational inactivation of the message (8, 9). Similar mechanisms seem to operate in neurons (10, 11) and possibly in other somatic cells (12).Because the length of the poly(A) tail is important for its function, it is not surprising that poly(A) tails are generally synthesized with a defined length, which is species-specific, ∼70–90 nucleotides in Saccharomyces cerevisiae (13, 14) and ∼250 nucleotides in mammalian cells (15). Subtle differences between newly made poly(A) tails of different mRNAs have been described (13), and there is even a class of mRNAs that never receives more than an oligo(A) tail (16, 17). However, the heterogeneous length distribution seen in the steady-state mRNA population is the result of cytoplasmic shortening starting from a relatively well defined initial tail length; heterogeneity of tail length reflects age differences of the mRNA molecules. The oligo(A) tails present on inactive mRNAs in oocytes or embryos are also generated by shortening of full-length tails made in the cell nucleus (18).The poly(A) tail is added during 3′ end processing of mRNA precursors in the cell nucleus (19–21). This reaction consists of two steps: an endonucleolytic cleavage followed by the addition of the poly(A) tail to the upstream cleavage product. Whereas a large protein machinery of some 20 or more polypeptides (22) is required for the cleavage reaction, subsequent polyadenylation has much simpler protein requirements. In the mammalian system, it can be reconstituted from three proteins: poly(A) polymerase, the enzyme catalyzing primer-dependent polymerization of AMP using ATP as a precursor (23–25); the cleavage and polyadenylation specificity factor (CPSF),⁶ which binds the cleavage and polyadenylation signal AAUAAA (26, 27); and the nuclear poly(A)-binding protein (PABPN1), which binds the growing poly(A) tail (28, 29). Note that PABPN1 is distinct from the family of cytoplasmic poly(A)-binding proteins (30). Roles of poly(A) polymerase and CPSF in polyadenylation in vivo have been most clearly demonstrated by genetic analysis of the orthologues in S. cerevisiae (21, 31). PABPN1 has no functional orthologue in budding yeast (32); its function in polyadenylation has been confirmed in mammalian cells (33) and in Drosophila (34).Whereas PABPN1 and poly(A) polymerase are monomeric proteins, CPSF is a hetero-oligomer, which has not yet been reconstituted from recombinant proteins (22, 26, 35–40). Poly(A) polymerase on its own is barely active because of a low affinity for its RNA substrate and thus acts distributively, i.e. it dissociates from the RNA after each polymerization step, and presumably often before it has incorporated any nucleotide; the enzyme also has no significant sequence specificity and will elongate any RNA with a free 3′ OH (24). Both CPSF and PABPN1 enhance the activity of the polymerase by recruiting the enzyme to its substrate through direct interactions (38, 41). Sequence specificity of poly(A) addition reflects the RNA binding specificities of the two stimulatory factors: CPSF recruits the polymerase to RNAs containing the AAUAAA sequence in the vicinity of their 3′ ends (24, 42, 43), and PABPN1 recruits the enzyme to substrate RNAs carrying a terminal oligo(A) tract (29). Each factor alone endows the polymerase with modest processivity, such that it can incorporate maybe two to five nucleotides before dissociating (44). RNAs containing both the AAUAAA sequence and an oligo(A) tail and thus resembling intermediates of the polyadenylation reaction support a cooperative or synergistic stimulation of poly(A) polymerase by both CPSF and PABPN1. Under these conditions, addition of the poly(A) tail occurs in a processive manner, i.e. without intermittent dissociation of the protein complex from its substrate RNA (29, 44).Interestingly, the reconstituted polyadenylation reaction also shows proper length control, generating poly(A) tails of the same length as seen in vivo; tails grow to a relatively well defined length of 250–300 nucleotides in a rapid, processive reaction (29, 44). Length control is due to termination of this processive elongation; extension beyond 250 A residues is largely distributive and therefore slow (45). These kinetics of in vitro poly(A) tail synthesis are fully consistent with the in vivo kinetics derived from pulse-labeling studies (46). In vitro, poly(A) tail elongation rates beyond 250 A residues are similar when either CPSF or PABPN1 or both are present. In other words, substrates with long poly(A) tails no longer support the cooperative stimulation of poly(A) polymerase by both CPSF and PABPN1 that is the basis of processive elongation (45). The termination of processive elongation must be mediated by a change in the RNA-protein complex that remains to be defined. When RNAs carrying poly(A) tails of different lengths are used as substrates for polyadenylation, the tails are always elongated processively to 250 nucleotides, independently of the initial length, whereas extension of a tail of 250 or more nucleotides in length is slow and distributive from the start of the reaction. Thus, poly(A) tail length control is based on some kind of AMP residue counting or length measurement, not on a kinetic mechanism (45).In this paper, we address the two problems outlined above: first, how does the polyadenylation complex change to terminate processive poly(A) tail elongation, and second, how is the length of the tail measured? We provide evidence that PABPN1 is the active component in the mechanism of length control. The protein promotes the interaction between CPSF and poly(A) polymerase when bound to a short poly(A) tail. PABPN1 no longer promotes or even actively disrupts this interaction when bound to a poly(A) tail of 250 nucleotides or longer and thereby terminates the cooperative, processive elongation reaction in a poly(A) tail length-dependent manner. Only poly(A) sequences are counted as part of the tail. Because this reflects the binding specificity of PABPN1 and because disruption of the CPSF-poly(A) polymerase interaction requires complete coverage of the poly(A) tail by this protein, PABPN1 is also the protein that measures the length of the tail.     

Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP

Summary

A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.

Results

We have selected a ssDNA aptamer population against a recombinant 6xHIS–LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.

Conclusion

Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.     

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.     

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Background

Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results

In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions

Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.     

An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.     

Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export

Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover. In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function. Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal. In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p. Here, we examine the nature of this lag-correction function. We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction. Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively. PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner. Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed. We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.     

Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis

The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is ‘masked’ shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca²⁺-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.     

Human PABP binds AU-rich RNA via RNA-binding domains 3 and 4.

Poly(A) binding protein (PABP) binds mRNA poly(A) tails and affects mRNA stability and translation. We show here that there is little free PABP in NIH3T3 cells, with the vast majority complexed with RNA. We found that PABP in NIH3T3 cytoplasmic lysates and recombinant human PABP can bind to AU-rich RNA with high affinity. Human PABP bound an AU-rich RNA with Kd in the nm range, which was only sixfold weaker than the affinity for oligo(A) RNA. Truncated PABP containing RNA recognition motif domains 3 and 4 retained binding to both AU-rich and oligo(A) RNA, whereas a truncated PABP containing RNA recognition motif domains 1 and 2 was highly selective for oligo(A) RNA. The inducible PABP, iPABP, was found to be even less discriminating than PABP in RNA binding, with affinities for AU-rich and oligo(A) RNAs differing by only twofold. These data suggest that iPABP and PABP may in some situations interact with other RNA regions in addition to the poly(A) tail.     

Poly(A)-binding proteins: Structure, domain organization, and activity regulation

RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA inter-actions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

The flip-flop configuration of the PABP-dimer leads to switching of the translation function

Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3′ end of an mRNA close to its 5′ m⁷G cap structure through consecutive interactions of the 3′-poly(A)–PABP-eIF4G-eIF4E-5′ m⁷G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP–PABP interaction. Poly(A) RNA was shown to convert the PABP–PABP complex into a poly(A)–PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)–PABP complex is necessary for the translational activation function.