poly(A)-binding Protein Research Articles

The short conserved region-2 of LARP4 interacts with ribosome-associated RACK1 and promotes translation.

LARP4 interacts with poly(A)-binding protein (PABP) to protect mRNAs from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved, and relevance had been undetermined. Here, yeast two-hybrid domain mapping followed by other methods identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and LARP4B) as directly binding RACK1 region 200-317. Consistent with these results, AlphaFold2-multimer predicted high confidence interaction of CR2 with RACK1 propellers 5-6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas less effect was observed for PABP association, consistent with independent interactions. CR2 mutations decreased LARP4 ability to optimally stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) more significantly than a β-globin and other reporters lacking this element. While polysome profiles indicate the β-glo-ARE mRNA is inefficiently translated, consistent with published data, we show that LARP4 increases its translation whereas the LARP4-CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.

MATE transporter OsMATE2 mediates root growth, grain size and weight by interacting with Mn-SOD and PABP in rice

Multidrug and toxic compound extrusion proteins (MATE) can transport small organic molecules in and out of cells and participate in detoxification, nutrient absorption, disease resistance and plant development processes. These compounds are widely distributed in plants. However, the mechanism by which MATE affects grain development remains elusive. In this study, we studied a MATE transporter, OsMATE2, which localized on the membrane. The CRISPR-Cas9 (CR) knockout line of OsMATE2 presented obvious decreases in grain weight. In addition, root development was also affected. Two proteins that interact with OsMATE2, namely, manganese-superoxide dismutase (Mn-SOD) and poly(A)-binding protein (PABP), were identified from a screening of yeast library. The results were validated through yeast two-hybrid and bimolecular fluorescence complementation experiments. The CRISPR-Cas9 (CR) knockout lines of Mn-SOD and PABP presented increased grain size and weight. Our findings demonstrated that OsMATE2 interacts with Mn-SOD and PABP to regulate grain development in rice.

Expansion and diversification of the Glycine max (Gm) ERD15-like subfamily of the PAM2-like superfamily.

Despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and diverge partially in stress signaling functions. The PAM2 motif represents a binding site for poly (A)-binding proteins (PABPs), often associated with RNA metabolism regulation. The PAM2-containing protein ERD15 stands out as a critical regulator of diverse stress responses in plants. Despite the relevance of the PAM2 motif, a comprehensive analysis of the PAM2 superfamily and ERD15-like subfamily in the plant kingdom is lacking. Here, we provide an extensive in silico analysis of the PAM2 superfamily and the ERD15-like subfamily in soybean, using Arabidopsis and rice sequences as prototypes. The Glycine max ERD15-like subfamily members were clustered in pairs, likely originating from DNA-based gene duplication, as the paralogs display high sequence conservation, similar exon/intron genome organization, and are undergoing purifying selection. Complementation analyses of an aterd15 mutant demonstrated that the plant ERD15-like subfamily members are functionally redundant in response to drought, osmotic stress, and dark-induced senescence. Nevertheless, the soybean members displayed differential expression profiles, biochemical activity, and subcellular localization, consistent with functional diversification. The expression profiles of Glyma04G138600 under salicylic acid (SA) and abscisic acid (ABA) treatments differed oppositely from those of the other GmERD15-like genes. Abiotic stress-induced coexpression analysis with soybean PABPs showed that Glyma04G138600 was clustered separately from other GmERD15s. In contrast to the AtERD15 stress-induced nuclear redistribution, Glyma04G138600 and Glyma02G260800 localized to the cytoplasm, while Glyma03G131900 fractionated between the cytoplasm and nucleus under normal and stress conditions. These data collectively indicate that despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and may diverge partially in stress signaling functions.

PABPN1 functions as a downstream gene of CREB to inhibit the proliferation of preadipocytes.

This study was designed to reveal the role of nuclear poly(A) binding protein 1 (PABPN1) in the proliferation of preadipocytes, and to reveal the relationship between PABPN1 and cAMP response element (CRE)-binding protein (CREB) in the regulation of preadipocyte proliferation. Vectors overexpressing and siRNAs against PABPN1/CREB were transiently transfected into both porcine preadipocytes and mouse 3T3-L1 cells. Preadipocyte proliferation was measured with CCK-8, EdU, real-time quantitative PCR, Western blotting, and flow cytometry analyses. Additionally, the transcriptional regulation of CREB on PABPN1 were analyzed with dual-luciferase reporter gene and EMSA assays. Overexpression of PABPN1 inhibits, and knockdown of PABPN1 promotes, the proliferation of both porcine preadipocytes and 3T3-L1 cell lines. PABPN1 overexpression increased, while knockdown decreased, the cell population in the G0/G1 phase. These indicates that PABPN1 repressed preadipocyte proliferation by inhibiting cell cycle progress. Additionally, it was revealed that CREB regulated the expression of PABPN1 through binding to the promoter and that CREB inhibited preadipocyte proliferation by repressed cell cycle progress. Furthermore, we showed that PABPN1 functions as a downstream gene of CREB to regulate the proliferation of preadipocytes. PABPN1 inhibits preadipocyte proliferation by suppressing the cell cycle. We also found that CREB could promote PABPN1 expression by binding to a motif in the promoter. Further analysis confirmed that PABPN1 functions as a downstream gene of CREB to regulate the proliferation of preadipocytes. These results suggest that the CREB/PABPN1 axis plays a role in the regulation of preadipocyte proliferation, which will contribute to further revealing the mechanism of fat accumulation.

Poised PABP–RNA hubs implement signal-dependent mRNA decay in development

Signaling pathways drive cell fate transitions largely by changing gene expression. However, the mechanisms for rapid and selective transcriptome rewiring in response to signaling cues remain elusive. Here we use deep learning to deconvolve both the sequence determinants and the trans-acting regulators that trigger extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase kinase (MEK)-induced decay of the naive pluripotency mRNAs. Timing of decay is coupled to embryo implantation through ERK–MEK phosphorylation of LIN28A, which repositions pLIN28A to the highly A+U-rich 3′ untranslated region (3′UTR) termini of naive pluripotency mRNAs. Interestingly, these A+U-rich 3′UTR termini serve as poly(A)-binding protein (PABP)-binding hubs, poised for signal-induced convergence with LIN28A. The multivalency of AUU motifs determines the efficacy of pLIN28A–PABP convergence, which enhances PABP 3′UTR binding, decreases the protection of poly(A) tails and activates mRNA decay to enable progression toward primed pluripotency. Thus, the signal-induced convergence of LIN28A with PABP–RNA hubs drives the rapid selection of naive mRNAs for decay, enabling the transcriptome remodeling that ensures swift developmental progression.

Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translationand decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.

Conformational Properties of Poly(A)-Binding Protein Complexed with Poly(A) RNA.

Microscopic understanding of protein-RNA interactions is important for different biological activities, such as RNA transport, translation, splicing, silencing, etc. Polyadenine (Poly(A)) binding proteins (PABPs) make up a class of regulatory proteins that play critical roles in protecting the poly(A) tails of cellular mRNAs from nuclease degradation. In this work, we performed molecular dynamics simulations to investigate the conformational modifications of human PABP protein and poly(A) RNA that occur during complexation. It is demonstrated that the intermediate linker domain of the protein transforms from a disordered coil-like structure to a helical form during the recognition process, leading to the formation of the complex. On the other hand, disordered collapsed coil-like RNA on complexation has been found to transform into a rigid extended conformation. Importantly, the binding free energy calculation showed that the thermodynamic stability of the complex is primarily guided by favorable hydrophobic interactions between the protein and the RNA.

Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Green-synthesized chitosan‑carbon dot nanocomposite as turn-on aptasensor for detection and quantification of Leishmania infantum parasite

Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94 cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188–750 cells/mL and 3000–6000 cells/mL (R2 ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.

PABPN1L is required for maternal mRNA degradation after meiosis resumption.

Poly(A)-binding proteins (PABPs) play roles in mRNA maturation, translational activity, and decay. The functions of PABPs, especially PABPN1 and PABPC1, in somatic cells have been well-studied. However, little is known about the roles of PABPs in oocytes because of the unique mechanisms of mRNA metabolism in oocytes. This study focused on PABPN1L and generated Pabpn1l knockout (KO) mice using the CRISPR/Cas9 system. After mating tests, we found that Pabpn1l KO females were infertile due to the failure of the embryos to develop to the 4-cell stage. RNA-seq analysis revealed aberrant mRNA persistence in Pabpn1l KO-MII oocytes, which indicates impaired mRNA degradation during the germinal vesicle (GV) to MII transition. We also revealed that the exogenous expression of Pabpn1l mRNA in KO-GV oocytes recovered defects of embryonic development. PABPN1L is partly indispensable for female fertility in mice, owing to its necessity for embryonic development, which is supported by mRNA degradation during GV to MII maturation.

Integrative Analysis of PAIP2B to Identify a Novel Biomarker for Pancreatic Ductal Adenocarcinoma.

The aim of the study was to evaluate the potential diagnostic and prognostic value of gene, Poly A-Binding Protein Interacting Protein 2B ( PAIP2B ) in pancreatic cancer. We used the gene expression data and clinical information of pancreatic adenocarcinoma patients from The Cancer Genome Atlas database and Gene Expression Omnibus database to analyze the expression of PAIP2B in pancreatic cancer samples, and validated the expression of PAIP2B in tumor tissue, using bioinformatics technology to explore the prognostic value of PAIP2B and its possible biological function. A significantly lower level of PAIP2B was observed in pancreatic cancer patients than in controls, and validated by immunohistochemistry. PAIP2B reduced the proliferation and invasion of cancer cells and had a significantly high expression in early stage. Patients with lower levels of PAIP2B had a significantly shorter median survival time than those with higher levels. DNA demethylation played an important role in PAIP2B expression. In addition, PAIP2B expression was significantly associated with the tumor-infiltrating immune cells, especially T cells CD8, T cells CD4 memory resting, macrophages M0, and dendritic cells resting. Our study also found that PAIP2B regulated miRNA function leading to disease progression in pancreatic cancer patients. Our study explored the potential value of PAIP2B as a biological link between prognosis and pancreatic cancer, and provided reference for the follow-up study on the role of PAIP2B in pancreatic cancer.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

Hyperglycemia facilitates dengue virus infection and host immune alteration

Abstract Patients with diabetes mellitus are highly comorbid with severe dengue diseases caused by the dengue virus (DENV). In search of possible host factors involved in DENV infection under hyperglycemic stress, cells and murine treated with high glucose (HG) increase viral protein expression and virion release but have no effects on antiviral interferon responses as well as viral early infectious stage. Following HG stimulation, a cellular replicon-based assay displays an increased viral translation while the glucose uptake inhibitor phloretin treatment blocks such an effect. Treating HG causes increases in the protein and gene expression of translational factor poly(A)-binding protein (PABP) in a glucose transporter-associated PI3K/AKT-regulated manner. PABP silence further causes a significant decrease in HG-increased virion production. In addition, HG treatment enhances the formation of the PABP-eukaryotic translation initiation factor 4G complex, regulated by protein-disulfide isomerase. Furthermore, streptozotocin-treated hyperglycemic mice reveal increased mortality, brain viral protein expression, and viral loads under DENV infection. This study demonstrates that hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation. This work is supported by the grants from the National Science and Technology Council (NSTC109-2327-B-006-010 and 110-2320-B-038-064-MY3), Taipei, Taiwan.

Diverse yeast antiviral systems prevent lethal pathogenesis caused by the L-A mycovirus

Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1; the SAGA and PAF1 chromatin regulatory complexes; and HSF1, the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.

Short poly(A) tails are protected from deadenylation by the LARP1-PABP complex.

Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.

Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis.

Background: During a developmental process, embryos employ varying tactics to remove unwanted cells. Using a procedure analogous to some of the embryonic cells, we generated a tumor-eliminating conditioned medium (CM) from AMPK-inhibited lymphocytes and monocytes in peripheral blood mononuclear cells (PBMCs). Methods: AMPK signaling was inhibited by the application of a pharmacological agent, Dorsomorphin, and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of mammary tumors and tumor-induced osteolysis. The regulatory mechanism was evaluated using mass spectrometry-based proteomics, Western blotting, immunoprecipitation, gene overexpression, and RNA interference. Results: While AMPK signaling acted mostly anti-tumorigenic, we paradoxically inhibited it to build induced tumor-suppressing cells and their tumor-eliminating CM. In a mouse model of breast cancer, the application of AMPK-inhibited lymphocyte-derived CM reduced mammary tumors additively to a chemotherapeutic agent, Taxol. It also prevented bone loss in the tumor-bearing tibia. Furthermore, the application of CM from the patient-derived peripheral blood diminished ex vivo breast cancer tissues isolated from the same patients. Notably, proteins enriched in CM included Moesin (MSN), Enolase 1 (ENO1), and polyA-binding protein 1 (PABPC1), which are considered tumorigenic in many types of cancer. The tumor-suppressing actions of MSN and ENO1 were at least in part mediated by Metadherin (Mtdh), which is known to promote metastatic seeding. Conclusion: We demonstrated that PBMCs can be used to generate tumor-suppressive proteomes, and extracellular tumor-suppressing proteins such as MSN, ENO1, and PABPC1 are converted from tumor-promoting factors inside cancer cells. The results support the possibility of developing autologous blood-based therapy, in which tumor-suppressing proteins are enriched in engineered PBMC-derived CM by the inhibition of AMPK signaling.

Birth of a poly(A) tail: mechanisms and control of mRNA polyadenylation.

During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3'-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3'-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3'-end processing.

Hyperglycemia exacerbates dengue virus infection by facilitating poly(A)-binding protein-mediated viral translation.

Diabetes mellitus (DM) is highly comorbid with severe dengue diseases; however, the underlying mechanisms are unclear. Patients with DM have a 1.61-fold increased risk of developing dengue hemorrhagic fever. In search of host factors involved in dengue virus (DENV) infection, we used high-glucose (HG) treatment and showed that HG increased viral protein expression and virion release but had no effects on the early stages of viral infection. After HG stimulation, DENV-firefly luciferase-transfected assay and cellular replicon-based assay indicated increased viral translation, whereas using the glucose uptake inhibitor phloretin blocked this effect. HG treatment increased the translational factor poly(A)-binding protein (PABP) in a glucose transporter-associated, PI3K/AKT-regulated manner. Silencing PABP significantly decreased HG-prompted virion production. HG enhanced the formation of the PABP-eukaryotic translation initiation factor 4G complex, which is regulated by protein-disulfide isomerase. Hyperglycemia increased PABP expression, mortality rate, viral protein expression, and viral loads in streptozotocin-induced DM mice. Overall, hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation.

Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells.

Flaviviruses are small RNA viruses that are mainly transmitted via arthropod vectors and are found in tropic and sub-tropical regions. Most infections are asymptomatic (90–95%), but symptoms can be as severe as hemorrhagic fever and encephalitis. One recently emerged flavivirus is Zika virus (ZIKV), which was originally isolated from rhesus monkeys in Uganda roughly 70 years ago but has recently spread east, reaching S. America in 2015–2016. This outbreak was associated with the development of Guillain–Barré syndrome in adults and microcephaly in infants born to expectant mothers infected early in pregnancy. ZIKV must traverse the placenta to impact the development of the fetus, but the mechanisms responsible are unknown. While flaviviruses are known to disrupt splicing patterns in host cells, little is known about how flaviviruses such as ZIKV impact the alternative polyadenylation (APA) of host transcripts. This is important as APA is well-established as a mechanism in the regulation of mRNA metabolism and translation. Thus, we sought to characterize transcriptomic changes including APA in human placental (JEG3) cells in response to ZIKV infection using Poly(A)-ClickSeq (PAC-Seq). We used our differential Poly(A)-cluster (DPAC) analysis pipeline to characterize changes in differential gene expression, alternative poly-adenylation (APA) and the use of alternative terminal exons. We identified 98 upregulated genes and 28 downregulated genes. Pathway enrichment analysis indicated that many RNA processing and immune pathways were upregulated in ZIKV-infected JEG3 cells. We also updated DPAC to provide additional metrics of APA including the percentage-distal usage index (PDUI), which revealed that APA was extensive and the 3′ UTRs of 229 genes were lengthened while 269 were shortened. We further found that there were 214 upregulated and 59 downregulated poly(A)-clusters (PACs). We extracted the nucleotide sequences surrounding these PACs and found that the canonical signals for poly-adenylation (binding site for poly-A binding protein (PABP) upstream and a GU-rich region down-stream of the PAC) were only enriched in the downregulated PACs. These results indicate that ZIKV infection makes JEG3 cells more permissive to non-canonical poly-adenylation signals.

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.