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.     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.     

Molecular Basis of eRF3 Recognition by the MLLE Domain of Poly(A)-Binding Protein

PABPC1 (cytosolic poly(A)-binding protein 1) is an RNA-binding protein that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. In addition to RNA-binding domains, PABPC1 contains a unique protein-protein interaction domain, MLLE (also known as PABC) that binds regulatory proteins and translation factors that contain a conserved 12 amino acid peptide motif termed PAM2. Eukaryotic Release Factor 3 (eRF3/GSPT1) contains two overlapping PAM2 sequences, which are required for its activity. Here, we determined the crystal structures of the MLLE domain from PABPC1 in complex with the two PAM2 regions of eRF3. The structures reveal a mechanism of cooperativity between the two PAM2 sites that increases the binding affinity but prevents the binding of more than one molecule of eRF3 to PABPC1. Relative to previous structures, the high-resolution crystal structures force a re-evaluation of the PAM2 motif and improve our understanding of the molecular basis of MLLE peptide recognition.     

Prediction of Lipid-Binding Regions in Cytoplasmic and Extracellular Loops of Membrane Proteins as Exemplified by Protein Translocation Membrane Proteins

The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation.     

Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

tRNAs and Proteins Are Imported into Mitochondria of Trypanosoma brucei by Two Distinct Mechanisms

Import of tRNA into the mitochondrial matrix of Trypanosoma brucei was reconstituted in vitro. Efficient import required the hydrolysis of externally added ATP and was shown to be a carrier-mediated process depending on proteinaceous receptors on the surface of mitochondria. A partly synthetic tRNA(Tyr) as well as a physiological tRNA(Lys) were imported along the same pathway. Contrary to import of all matrix-localized proteins, tRNA import does not require a membrane potential. Furthermore, addition of an excess of import-competent tRNA had no effect on import of a mitochondrial matrix protein. In summary, these results show that tRNAs and proteins in T. brucei are imported by fundamentally different mechanisms.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.     

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.     

Early Events of Fusion Between Epstein Barr Virus and Human Lymphoblastoid Cells (Raji) Detected by R18 Fluorescence Dequenching Measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promoter and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vescicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.     

Poly(A)-Binding Protein 1 Partially Relocalizes to the Nucleus during Herpes Simplex Virus Type 1 Infection in an ICP27-Independent Manner and Does Not Inhibit Virus Replication

Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection.The lytic replication cycle of herpes simplex virus type 1 (HSV-1) can be divided into three phases, immediate-early (IE), early (E), and late (L), that occur in a coordinated sequential gene expression program. IE proteins can regulate E and L gene expression, which produces proteins involved in DNA replication, capsid production, and virion assembly. HSV infection results in host cell shutoff to facilitate the efficient production of viral proteins. First, mRNA is degraded by the virion-associated vhs protein, and then ICP27, a multifunctional regulator of gene expression, inhibits pre-mRNA splicing. As most viral mRNAs are intronless, this abrogates the production of stable cellular mRNAs that can be exported to the cytoplasm and compete for translation with viral mRNAs (44).HSV mRNAs are capped and polyadenylated and so are translated via a normal cap-dependent mechanism. Translation initiation, during which translationally active ribosomes are assembled, is a tightly regulated process (21). Eukaryotic initiation factor 4F (eIF4F) (composed of eIF4E, eIF4G, and eIF4A) that binds the cap at the 5′ end of the mRNA promotes the recruitment of the 40S ribosomal subunit and associated factors, including eIF2-GTP initiator tRNA. The recognition of the start codon then promotes large ribosomal subunit joining. Poly(A)-binding protein 1 (PABP1), which binds and multimerizes on mRNA poly(A) tails, enhances translation initiation through interactions with the eIF4G component of the eIF4F cap-binding complex (20, 29, 32, 51) to circularize the mRNA in a “closed-loop” conformation (24). Key protein-RNA and protein-protein interactions in the translation initiation complex are strengthened by this PABP1-mediated circularization (12).HSV-1 maintains active viral translation in the face of host translational shutoff. Infection activates protein kinase R (PKR), which phosphorylates eIF2α, resulting in translation inhibition. However, HSV-1 ICP34.5 redirects protein phosphatase 1α to reverse eIF2α phosphorylation, abrogating the block to translation (17, 38). In addition, the HSV-1 US11 protein inhibits PKR and may also block PKR-mediated eIF2α phosphorylation (40, 42). HSV-1 infection also enhances eIF4F assembly in quiescent cells by the phosphorylation and proteasome-mediated degradation of the eIF4E-binding protein (4E-BP), which, when hypophosphorylated, can negatively regulate eIF4F complex formation (54). However, ICP6 may also contribute to eIF4F assembly by binding to eIF4G (55). Finally, ICP6 is required for Mnk-1 phosphorylation of eIF4E, but the mechanisms behind this remain unclear (54). ICP27 has also been implicated in translation regulation during HSV infection (6, 8, 10, 30) and may also activate p38 mitogen-activated protein (MAP) kinase that can phosphorylate eIF4E (16, 59).PABP1 appears to be a common cellular target of RNA and DNA viruses. PABP1 can undergo proteolysis, intracellular relocalization, or modification of its interaction with other translation factors in response to infection. For example, poliovirus induces host cell shutoff by cleaving PABP1, thus disrupting certain PABP1-containing complexes (28, 29). The rotavirus NSP3 protein can displace PABP1 from translation initiation complexes (41). However, NSP3 also interacts with a cellular protein, RoXaN, which is required to relocate PABP1 to the nucleus (13). Similarly, the Kaposi''s sarcoma herpesvirus (KSHV) SOX protein plays a role in relocating PABP1, its cofactor in cellular mRNA decay, to the nucleus (33). Although steady-state levels of PABP1 are highest in the cytoplasm of normal cells, where it has cytoplasmic functions, it is a nucleocytoplasmic shuttling protein (1). However, it is unclear how PABP1 enters or exits the nucleus, as it contains neither a canonical nuclear export nor an import signal.Here we describe the loss of PABP1 from cap-binding complexes and the partial relocation of PABP1 to the nucleus in HSV-1-infected cells in a time-dependent manner. Relocation is specific for PABP1, as other translation factors remained in the cytoplasm. Cells undergo stress during HSV-1 infection, and analysis of a variety of cell stresses revealed that PABP relocalization was also observed upon oxidative stress. Paxillin, a potential PABP1 nuclear chaperone, was phosphorylated, and the paxillin-PABP1 interaction was reduced during virus infection. However, the interaction was weak and cell type dependent, indicating that other effectors of PABP1 relocation in the infected cell must exist. Recently, the HSV-1 ICP27 protein was suggested to alter the PABP1 cellular location (6). However, infections with ICP27-null mutant viruses clearly demonstrated that ICP27 is not required for PABP1 nuclear relocation in the context of infection. Although HSV-1 mRNAs are translated by a normal cap-dependent mechanism known to be enhanced by PABP1, small interfering RNA (siRNA) knockdown of PABP1 indicated that at late times of infection, the translation of certain virus late proteins tolerates very low levels of PABP1.