PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity.

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

PABP prevents the untimely decay of select mRNA populations in human cells.

Gene expression is tightly regulated at the levels of both mRNA translation and stability. The poly(A)-binding protein (PABP) is thought to play a role in regulating these processes by binding the mRNA 3' poly(A) tail and interacting with both the translation and mRNA deadenylation machineries. In this study, we directly investigate the impact of PABP on translation and stability of endogenous mRNAs in human cells. Remarkably, our transcriptome-wide analysis only detects marginal mRNA translation changes in PABP-depleted cells. In contrast, rapidly depleting PABP alters mRNA abundance and stability, albeit non-uniformly. Otherwise stable transcripts, including those encoding proteins with constitutive functions, are destabilized in PABP-depleted cells. In contrast, many unstable mRNAs, including those encoding proteins with regulatory functions, decay at similar rates in presence or absence of PABP. Moreover, PABP depletion-induced cell death can partially be suppressed by disrupting the mRNA decapping and 5'-3' decay machinery. Finally, we provide evidence that the LSM1-7 complex promotes decay of "stable" mRNAs in PABP-depleted cells. Taken together, these findings suggest that PABP plays an important role in preventing the untimely decay of select mRNA populations.

A YTHDF-PABP interaction is required for m6 A-mediated organogenesis in plants.

N6-methyladenosine (m6 A) in mRNA is key to eukaryotic gene regulation. Many m6 A functions involve RNA-binding proteins that recognize m6 A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3, and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR elements required for ECT2-mediated organogenesis. Of these two, a 19-amino acid region containing a tyrosine-rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)-binding proteins PAB2, PAB4, and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells.

I-KCKT allows dissection-free RNA profiling of adult Drosophila intestinal progenitor cells.

The adult Drosophila intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed intestinal-kickout-GAL4 (I-KCKT-GAL4), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by I-KCKT-GAL4 When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.8% of transcripts found after progenitor cell sorting, and has low background noise despite being derived from whole animal lysate. We also mapped the targets of the more selective RNA binder, Fragile X mental retardation protein (FMRP), using enhanced crosslinking and immunoprecipitation (eCLIP), and report for the first time its binding motif in Drosophila cells. This method will therefore enable the RNA profiling of wild-type and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions crucial for stem cell function.

Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail.

Eukaryotic mRNAs possess a poly(A) tail at their 3'-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1-poly(A) and PABPC1-Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2-RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, Kd = 1 nM). However, the Kd values of isolated RRM2 were 200 and 4 µM in their interactions with poly(A) and Paip2A, respectively; Kd values of 5 and 1 µM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.

When Poly(A) Binding Proteins Meet Viral Infections, Including SARS-CoV-2.

Viruses have evolved diverse strategies to hijack the cellular gene expression system for their replication. The poly(A) binding proteins (PABPs), a family of critical gene expression factors, are viruses' common targets. PABPs act not only as a translation factor but also as a key factor of mRNA metabolism. During viral infections, the activities of PABPs are manipulated by various viruses, subverting the host translation machinery or evading the cellular antiviral defense mechanism. Viruses harness PABPs by modifying their stability, complex formation with other translation initiation factors, or subcellular localization to promote viral mRNAs translation while shutting off or competing with host protein synthesis. For the past decade, many studies have demonstrated the PABPs' roles during viral infection. This review summarizes a comprehensive perspective of PABPs' roles during viral infection and how viruses evade host antiviral defense through the manipulations of PABPs.

Single polysome analysis of mRNP.

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation.

Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation.

Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover, it was unclear whether Tob2 phosphorylation modulates the transcriptome and whether the phosphorylation is linked to Tob2's anti-proliferative function. In this study, we found that c-Jun amino-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2-PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution profiling, transcriptome-wide mRNA turnover analyses, and cell proliferation assays, we found that the phosphomimetic mutation at S254 (S254D) enhances Tob2's association with PABP, leading to accelerated deadenylation and decay of mRNAs globally. Moreover, the Tob2-S254D mutant accelerates the decay of many transcripts coding for cell cycle related proteins and enhances anti-proliferation function. Our findings reveal a novel mechanism by which Ccr4-Not complex is recruited by Tob2 to the mRNA 3' poly(A)-PABP complex in a phosphorylation dependent manner to promote rapid deadenylation and decay across the transcriptome, eliciting transcriptome reprogramming and suppressed cell proliferation.

Chemically Modified Poly(A) Analogs Targeting PABP: Structure Activity Relationship and Translation Inhibitory Properties.

Poly(A)-binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate-derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein-RNA and protein-protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis-based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA-based biological tools.

[Dynamics of Eukaryotic mRNA Structure during Translation].

Currently, there is no single concept of the optimal spatial structure of mRNA during translation. It is known that many proteins, associated with the 5' end of mRNA, interact with proteins associated with the 3' end. Moreover, this interaction often affects the activity of these proteins. It is possible within the same mRNA molecule only when the mRNA forms a circular structure in which its ends are spatially close. Discovery of such proteins, in the 90s of the 20th century, made it possible to formulate the closed-loop mRNA structure hypothesis, in which it is assumed that the ends of translationally active mRNA are fixed next to each other due to the interaction of proteins and (or) RNA. However, later it was shown that a closed-loop structure is not always necessary for translation. Moreover, some authors have proposed a model according to which the translating mRNA, on the contrary, should be unfolded into a linear structure. Thus, the spatial structure of the translating mRNA does not have to be universal for all mRNA and can change dynamically, which affects its functional activity. In this review, we have summarized a variety of experimental data and concepts on the relationship between the spatial structure of mRNA and its translational activity.

[Role of Proteins Interacting with the eRF1 and eRF3 Release Factors in the Regulation of Translation and Prionization].

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

LARP1 and LARP4: up close with PABP for mRNA 3' poly(A) protection and stabilization.

La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3'OH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3' end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3' oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3' poly(A) tails from deadenylases through close contact with PABP.Abbreviations: 5'TOP: 5' terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.

Controversies around the function of LARP1.

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.

A new role for the cellular PABP repressor Paip2 as an innate restriction factor capable of limiting productive cytomegalovirus replication.

The capacity of polyadenylate-binding protein PABPC1 (PABP1) to stimulate translation is regulated by its repressor, Paip2. Paradoxically, while PABP accumulation promotes human cytomegalovirus (HCMV) protein synthesis, we show that this is accompanied by an analogous increase in the abundance of Paip2 and EDD1, an E3 ubiquitin ligase that destabilizes Paip2. Coordinate control of PABP1, Paip2, and EDD1 required the virus-encoded UL38 mTORC1 activator and resulted in augmented Paip2 synthesis, stability, and association with PABP1. Paip2 synthesis also increased following serum stimulation of uninfected normal fibroblasts, suggesting that this coregulation may play a role in how uninfected cells respond to stress. Significantly, Paip2 accumulation was dependent on PABP accrual, as preventing PABP1 accumulation suppressed viral replication and inhibited the corresponding Paip2 increase. Furthermore, depleting Paip2 restored the ability of infected cells to assemble the translation initiation factor eIF4F, promoting viral protein synthesis and replication without increasing PABP1. This establishes a new role for the cellular PABP1 inhibitor Paip2 as an innate defense that restricts viral protein synthesis and replication. Moreover, it illustrates how a stress-induced rise in PABP1 triggered by virus infection can counter and surpass a corresponding increase in Paip2 abundance and stability.

Characterization of ALBA Family Expression and Localization in Arabidopsis thaliana Generative Organs.

ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.

Musashi interaction with poly(A)-binding protein is required for activation of target mRNA translation.

The Musashi family of mRNA translational regulators controls both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed MS to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A)-binding protein (ePABP) or the canonical somatic cell poly(A)-binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate.

Polyadenylate-binding protein-interacting proteins PAIP1 and PAIP2 affect translation termination.

Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.

Translation of insulin granule proteins are regulated by PDI and PABP.

Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.

Rotavirus Infection Alters Splicing of the Stress-Related Transcription Factor XBP1.

XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA (XBP1es) that corresponded to exon skipping in the XBP1 pre-RNA is induced depending on the rotavirus strain used. We show that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using monoreassortant between ES+ ("skipping") and ES- ("nonskipping") strains of rotavirus, we show that gene 7 encoding the viral translation enhancer NSP3 is involved in this phenomenon and that exon skipping parallels the nuclear relocalization of cytoplasmic PABP. We further show, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype is linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP.IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA translation enhancer NSP3. Since XBP1 is involved in cellular stress and immune responses and since the XBP1 protein made from the alternatively spliced RNA is an active transcription factor, our observations raise the question of whether alternative splicing is a cellular response to rotavirus infection.