Solution structure of the orphan PABC domain from Saccharomyces cerevisiae poly(A)-binding protein

We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae Pab1p and mapped its peptide-binding site. PABC domains are peptide binding domains found in poly(A)-binding proteins (PABP) and are a subset of HECT-family E3 ubiquitin ligases (also known as hyperplastic discs proteins (HYDs)). In mammals, the PABC domain of PABP functions to recruit several different translation factors to the mRNA poly(A) tail. PABC domains are highly conserved, with high specificity for peptide sequences of roughly 12 residues with conserved alanine, phenylalanine, and proline residues at positions 7, 10, and 12. Compared with human PABP, the yeast PABC domain is missing the first alpha helix, contains two extra amino acids between helices 2 and 3, and has a strongly bent C-terminal helix. These give rise to unique peptide binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1. Mapping of the peptide-binding site reveals that the bend in the C-terminal helix disrupts binding interactions with the N terminus of peptide ligands and leads to greatly reduced binding affinity for the peptides tested. No high affinity or natural binding partners from S. cerevisiae could be identified by sequence analysis of known PABC ligands. Comparison of the three known PABC structures shows that the features responsible for peptide binding are highly conserved and responsible for the distinct but overlapping binding specificities.     

Solution structure of the PABC domain from wheat poly (A)-binding protein: an insight into RNA metabolic and translational control in plants

In animals, the PABC domain from poly (A)-binding protein recruits proteins containing a specific interacting motif (PAM-2) to the mRNP complex. These proteins include Paip1, Paip2, and eukaryotic release factor 3 (eRF3), all of which regulate PABP function in translation. The following reports the solution structure of PABC from Triticum avestium (wheat) poly (A)-binding protein determined by NMR spectroscopy. Wheat PABC (wPABC) is an alpha-helical protein domain, which displays a fold highly similar to the human PABC domain and contains a PAM-2 peptide binding site. Through a bioinformatics search, several plant proteins containing a PAM-2 site were identified including the early response to dehydration protein (ERD-15), which was previously shown to regulate PABP-dependent translation. The plant PAM-2 proteins contain a variety of conserved sequences including a PABP-interacting 1 motif (PAM-1), RNA binding domains, an SMR endonuclease domain, and a poly (A)-nuclease regulatory domain, all of which suggest a function in either translation or mRNA metabolism. The proteins identified are well conserved throughout plant species but have no sequence homologues in metazoans. We show that wPABC binds to the plant PAM-2 motif with high affinity through a conserved mechanism. Overall, our results suggest that plant species have evolved a distinct regulatory mechanism involving novel PABP binding partners.     

Poly(A) nuclease interacts with the C-terminal domain of polyadenylate-binding protein domain from poly(A)-binding protein

The poly(A)-binding protein (PABP) is an essential protein found in all eukaryotes and is involved in an extensive range of cellular functions, including translation, mRNA metabolism, and mRNA export. Its C-terminal region contains a peptide-interacting PABC domain that recruits proteins containing a highly specific PAM-2 sequence motif to the messenger ribonucleoprotein complex. In humans, these proteins, including Paip1, Paip2, eRF3 (eukaryotic release factor 3), Ataxin-2, and Tob2, are all found to regulate translation through varying mechanisms. The following reports poly(A) nuclease (PAN) as a PABC-interacting partner in both yeast and humans. Their interaction is mediated by a PAM-2 motif identified within the PAN3 subunit. This site was identified in various fungal and animal species suggesting that the interaction is conserved throughout evolution. Our results indicate that PABP is directly involved in recruiting a deadenylase to the messenger ribonucleoprotein complex. This demonstrates a novel role for the PABC domain in mRNA metabolic processes and gives further insight into the function of PABP in mRNA maturation, export, and turnover.     

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.     

Comparative peptide binding studies of the PABC domains from the ubiquitin-protein isopeptide ligase HYD and poly(A)-binding protein. Implications for HYD function

The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.     

Crystal structure of the middle domain of human poly(A)-binding protein-interacting protein 1

In eukaryotes, the poly(A)-binding protein (PABP) is one of the important factors for initiation of messenger RNA translation. PABP activity is regulated by the PABP-interacting proteins (Paips), which include Paip1, Paip2A, and Paip2B. Human Paip1 has three different isoforms. Here, we report the crystal structure of the middle domain of Paip1 isoform 2 (Paip1M) as determined by single-wavelength anomalous dispersion phasing. The structure reveals a crescent-shaped domain consisting of 10 α-helices and two antiparallel β-strands forming a β-hairpin. The 10 α-helices are arranged as five HEAT repeats which form a double layer of α helices with a convex and a concave surface. Despite low sequence identity, the overall fold of Paip1M is similar to the middle domain of human eIF4GII and yeast eIF4GI. Moreover, the amino-acid sequence motif and the local structure of eIF4G involved in binding of eIF4A, are conserved in Paip1. The structure reported here is the first of a member of the Paip family, thereby filling a gap in our understanding of initiation of eukaryotic mRNA translation in three dimensions.     

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.     

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independent K(d)s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.     

Regulation of poly(A) binding protein function in translation: Characterization of the Paip2 homolog, Paip2B

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This synergy is mediated via interactions between eIF4G (a component of the eIF4F cap binding complex) and poly(A) binding protein (PABP). Paip2 (PABP-interacting protein 2) binds PABP and inhibits translation both in vitro and in vivo by decreasing the affinity of PABP for polyadenylated RNA. Here, we describe the functional characteristics of Paip2B, a Paip2 homolog. A full-length brain cDNA of Paip2B encodes a protein that shares 59% identity and 80% similarity with Paip2 (Paip2A), with the highest conservation in the two PABP binding domains. Paip2B acts in a manner similar to Paip2A to inhibit translation of capped and polyadenylated mRNAs both in vitro and in vivo by displacing PABP from the poly(A) tail. Also, similar to Paip2A, Paip2B does not affect the translation mediated by the internal ribosome entry site (IRES) of hepatitis C virus (HCV). However, Paip2A and Paip2B differ with respect to both mRNA and protein distribution in different tissues and cell lines. Paip2A is more highly ubiquitinated than is Paip2B and is degraded more rapidly by the proteasome. Paip2 protein degradation may constitute a primary mechanism by which cells regulate PABP activity in translation.     

Translational repression by a novel partner of human poly(A) binding protein, Paip2

The eukaryotic mRNA 3' poly(A) tail acts synergistically with the 5' cap structure to enhance translation. This effect is mediated by a bridging complex, composed of the poly(A) binding protein (PABP), eIF4G, and the cap binding protein, eIF4E. PABP-interacting protein 1 (Paip1) is another factor that interacts with PABP to coactivate translation. Here, we describe a novel human PABP-interacting protein (Paip2), which acts as a repressor of translation both in vitro and in vivo. Paip2 preferentially inhibits translation of a poly(A)-containing mRNA, but has no effect on the translation of hepatitis C virus mRNA, which is cap- and eIF4G-independent. Paip2 decreases the affinity of PABP for polyadenylate RNA, and disrupts the repeating structure of poly(A) ribonucleoprotein. Furthermore, Paip2 competes with Paip1 for PABP binding. Thus, Paip2 inhibits translation by interdicting PABP function.     

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.     

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.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

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.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability

The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ～150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.     

Survey on the PABC recognition motif PAM2

The PABP-interacting motif PAM2 has been identified in various eukaryotic proteins as an important binding site for the PABC domain. This domain is contained in homologs of the poly(A)-binding protein PABP and the ubiquitin-protein ligase HYD. Despite the importance of the PAM2 motif, a comprehensive analysis of its occurrence in different proteins has been missing. Using iterated sequence profile searches, we obtained an extensive list of proteins carrying the PAM2 motif. We discuss their functional context and domain architecture, which often consists of RNA-binding domains. Our list of PAM2 motif proteins includes eukaryotic homologs of eRF3/GSPT1/2, PAIP1/2, Tob1/2, Ataxin-2, RBP37, RBP1, Blackjack, HELZ, TPRD, USP10, ERD15, C1D4.14, and the viral protease P29. The identification of the PAM2 motif in as yet uncharacterized proteins can give valuable hints with respect to their cellular function and potential interaction partners and suggests further experimentation. It is also striking that the PAM2 motif appears to occur solely outside globular protein domains.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.