ABSTRACT
Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges.
Subject(s)
Amino Acids , Eukaryotic Initiation Factor-4E , Gene Expression Regulation, Fungal , Protein Biosynthesis , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4E/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Amino Acids/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , 5' Untranslated Regions , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Cyclins/genetics , Cyclins/metabolism , Poly(A)-Binding Proteins/metabolism , Poly(A)-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
Ataxin-2 , Drosophila Proteins , Drosophila melanogaster , RNA, Messenger , Ribonucleoproteins , Ataxin-2/genetics , Ataxin-2/metabolism , Animals , Humans , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Poly(A)-Binding Proteins/metabolism , Poly(A)-Binding Proteins/genetics , Animals, Genetically Modified , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Protein Biosynthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , DNA-Binding ProteinsABSTRACT
Cells must sense and respond to sudden maladaptive environmental changes-stresses-to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide a remarkable view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein-a core marker of stress granules-condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry.
Subject(s)
Heat-Shock Response , Stress, Physiological , Heat-Shock Response/genetics , Stress, Physiological/genetics , Biological Evolution , Poly(A)-Binding Proteins/geneticsABSTRACT
Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.
Subject(s)
Heat-Shock Proteins , Poly(A)-Binding Proteins , Poly(A)-Binding Proteins/genetics , Temperature , Heat-Shock Proteins/metabolism , Thermodynamics , Heat-Shock Response , Deuterium Exchange Measurement/methodsABSTRACT
During maternal-to-zygotic transition (MZT) in the embryo, mRNA undergoes complex post-transcriptional regulatory processes. However, it is unclear whether and how alternative splicing plays a functional role in MZT. By analyzing transcriptome changes in mouse and human early embryos, dynamic changes in alternative splicing during MZT are observed and a previously unnoticed process of zygotic splicing activation (ZSA) following embryonic transcriptional activation is described. As the underlying mechanism of RNA splicing, splicing factors undergo dramatic maternal-to-zygotic conversion. This conversion relies on the key maternal factors BTG4 and PABPN1L and is zygotic-transcription-dependent. CDK11-dependent phosphorylation of the key splicing factor, SF3B1, and its aggregation with SRSF2 in the subnuclear domains of 2-cell embryos are prerequisites for ZSA. Isoforms generated by erroneous splicing, such as full-length Dppa4, hinder normal embryonic development. Moreover, alternative splicing regulates the conversion of early embryonic blastomeres from totipotency to pluripotency, thereby affecting embryonic lineage differentiation. ZSA is an essential post-transcriptional process of MZT and has physiological significance in generating new life. In addition to transcriptional activation, appropriate expression of transcript isoforms is also necessary for preimplantation embryonic development.
Subject(s)
Transcriptome , Zygote , Humans , Animals , Mice , Transcriptome/genetics , Zygote/metabolism , Embryonic Development/genetics , RNA Splicing , Protein Isoforms/genetics , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Nuclear Proteins/geneticsABSTRACT
Besides ubiquitous poly(A)-binding protein, cytoplasmic 1 (PABPC1), testis-specific PABPC2/PABPt (in humans, referred to as PABPC3), and female and male germline-specific PABPC1L/ePAB, have been reported in the mouse testis. Recent in silico analysis additionally identified testis-specific Pabpc6 in the mouse. In this study, we characterized PABPC6 and its mutant mice. PABPC6 was initially detectable in the cytoplasm of pachytene spermatocytes, increased in abundance in round spermatids, and decreased in elongating spermatids. PABPC6 was capable of binding to poly(A) tails of various mRNAs and interacting with translation-associated factors, including EIF4G, PAIP1, and PAIP2. Noteworthy was that PABPC6, unlike PABPC1, was barely associated with translationally active polysomes and enriched in chromatoid bodies of round spermatids. Despite these unique characteristics, neither synthesis of testicular proteins nor spermatogenesis was affected in the mutant mice lacking PABPC6, suggesting that PABPC6 is functionally redundant with other co-existing PABPC proteins during spermatogenesis.
Subject(s)
Spermatogenesis , Testis , Humans , Male , Mice , Female , Animals , Testis/metabolism , Spermatogenesis/genetics , Spermatids/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Cytoplasm/metabolism , RNA, Messenger/metabolism , Peptide Initiation Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Age-associated hypercoagulability is accompanied by the increase of plasma levels of some coagulation factors including fibrinogen which may contribute to the increased risk of cardiovascular, cerebrovascular, and thrombotic diseases in elderly people. However, the underlying mechanism of increased plasma fibrinogen concentration during aging is still elusive. GRSF1 belongs to the heterogeneous nuclear ribonucleoproteins F/H (hnRNP F/H) subfamily. Here, we report that GRSF1 attenuates hypercoagulability via negative modulation of fibrinogen expression. We demonstrated that GRSF1 negatively regulated fibrinogen expression at both mRNA and protein levels. GRSF1 directly interacted with the coding region (CDS) of FGA, FGB, and FGG mRNAs, and decreased their stability thus mitigating fibrinogen expression. We further identified that only a few G-tracts within the Fib C domain of FGA, FGB, and FGG CDS and the qRRM2 domain of GRSF1 were required for their interaction. Moreover, we confirmed hypercoagulability and the decrease of GRSF1 expression level during mice aging. Functionally, GRSF1 overexpression in old mice liver decreased fibrinogen plasma level, reduced hypercoagulability, and mitigated blood coagulation activity, whereas GRSF1 knockdown in young mice liver increased fibrinogen plasma level and promoted blood coagulation activity. Collectively, our findings unveil a novel posttranscriptional regulation of fibrinogen by GRSF1 and uncover a critical role of GRSF1 in regulating blood coagulation activity.
Subject(s)
Fibrinogen , Thrombophilia , Aged , Animals , Humans , Mice , Fibrinogen/genetics , Fibrinogen/metabolism , Gene Expression Regulation , Poly(A)-Binding Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , RNA-Binding Proteins/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , RNA, Messenger/genetics , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , RNA, Messenger/genetics , RNA Transport , Transcription, Genetic , Nucleocytoplasmic Transport Proteins/genetics , Poly(A)-Binding Proteins/geneticsABSTRACT
The mRNA 3' poly(A) tail plays a critical role in regulating both mRNA translation and turnover. It is bound by the cytoplasmic poly(A) binding protein (PABPC), an evolutionarily conserved protein that can interact with translation factors and mRNA decay machineries to regulate gene expression. Mammalian PABPC1, the prototypical PABPC, is expressed in most tissues and interacts with eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation in specific contexts. In this study, we uncovered a new mammalian PABPC, which we named neural PABP (neuPABP), as it is predominantly expressed in the brain. neuPABP maintains a unique architecture as compared with other PABPCs, containing only two RNA recognition motifs (RRMs) and maintaining a unique N-terminal domain of unknown function. neuPABP expression is activated in neurons as they mature during synaptogenesis, where neuPABP localizes to the soma and postsynaptic densities. neuPABP interacts with the noncoding RNA BC1, as well as mRNAs coding for ribosomal and mitochondrial proteins. However, in contrast to PABPC1, neuPABP does not associate with actively translating mRNAs in the brain. In keeping with this, we show that neuPABP has evolved such that it does not bind eIF4G and as a result fails to support protein synthesis in vitro. Taken together, these results indicate that mammals have expanded their PABPC repertoire in the brain and propose that neuPABP may support the translational repression of select mRNAs.
Subject(s)
Eukaryotic Initiation Factor-4G , Poly(A)-Binding Proteins , Animals , Poly(A)-Binding Proteins/genetics , Neurons , Brain , MammalsABSTRACT
BACKGROUND: RNA N6-methyladenosine (m6A) modification is critical for plant growth and crop yield. m6A reader proteins can recognize m6A modifications to facilitate the functions of m6A in gene regulation. ECT2, ECT3, and ECT4 are m6A readers that are known to redundantly regulate trichome branching and leaf growth, but their molecular functions remain unclear. RESULTS: Here, we show that ECT2, ECT3, and ECT4 directly interact with each other in the cytoplasm and perform genetically redundant functions in abscisic acid (ABA) response regulation during seed germination and post-germination growth. We reveal that ECT2/ECT3/ECT4 promote the stabilization of their targeted m6A-modified mRNAs, but have no function in alternative polyadenylation and translation. We find that ECT2 directly interacts with the poly(A) binding proteins, PAB2 and PAB4, and maintains the stabilization of m6A-modified mRNAs. Disruption of ECT2/ECT3/ECT4 destabilizes mRNAs of ABA signaling-related genes, thereby promoting the accumulation of ABI5 and leading to ABA hypersensitivity. CONCLUSION: Our study reveals a unified functional model of m6A mediated by m6A readers in plants. In this model, ECT2/ECT3/ECT4 promote stabilization of their target mRNAs in the cytoplasm.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Abscisic Acid , Germination/genetics , RNA Stability , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Seeds/genetics , Gene Expression Regulation, Plant , Intracellular Signaling Peptides and Proteins/geneticsABSTRACT
Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.
Subject(s)
Serine , Tyrosine , Humans , Animals , Mice , Phosphorylation , Tyrosine/metabolism , Serine/metabolism , Threonine/metabolism , Oocytes , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
Exoribonucleases , RNA-Binding Proteins , Exoribonucleases/metabolism , RNA-Binding Proteins/metabolism , Poly(A)-Binding Proteins/genetics , Gene Expression Regulation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Poly A/metabolismABSTRACT
Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and genetic techniques have revealed the fundamental mechanism that determines what occurs and when, where and in what order. Most mRNAs are circularized via the eIF4E-eIF4G-PABP interaction, which stabilizes mRNAs and enhances translation by recycling ribosomes. However, studies using single-molecule fluorescence imaging have allowed for the visualization of complex data that opposes the traditional "functional circularization" theory. Here, we briefly introduce single-molecule techniques applied to studies on mRNA circularization and describe the results of in vitro and live-cell imaging. Finally, we discuss relevant insights and questions gained from single-molecule research related to translation.
Subject(s)
Poly(A)-Binding Proteins , Protein Biosynthesis , RNA, Messenger/metabolism , Poly(A)-Binding Proteins/chemistry , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Protein Binding , Eukaryotic Initiation Factor-4G/chemistry , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolismABSTRACT
Under environmental stress, such as glucose deprivation, cells form stress granules-the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative diseases, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. An important aspect of stress granule formation is the presence of posttranslational modifications on core proteins. One of those modifications is lysine acetylation, which is regulated by either a lysine acetyltransferase or a lysine deacetylase enzyme. This work deciphers the impact of lysine acetylation on an essential protein found in Saccharomyces cerevisiae stress granules, poly(A)-binding protein (Pab1). We demonstrated that an acetylation mimic of the lysine residue in position 131 reduces stress granule formation upon glucose deprivation and other stressors such as ethanol, raffinose, and vanillin. We present genetic evidence that the enzyme Rpd3 is the primary candidate for the deacetylation of Pab1-K131. Further, our electromobility shift assay studies suggest that the acetylation of Pab1-K131 negatively impacts poly(A) RNA binding. Due to the conserved nature of stress granules, therapeutics targeting the activity of lysine acetyltransferases and lysine deacetylase enzymes may be a promising route to modulate stress granule dynamics in the disease state.
Subject(s)
Poly(A)-Binding Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Stress Granules , Acetylation , Glucose/metabolism , Lysine/metabolism , Lysine Acetyltransferases/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
PURPOSE: We aimed to identify pathogenic variants in a female patient with primary infertility and recurrent failure of in vitro fertilization with zygotic cleavage failure. METHODS: The genomic DNA from the affected individual was subjected to whole-exome sequencing and the variant was confirmed by Sanger sequencing. The functional effect of the identified variant was further investigated in 293 T cells. RESULTS: We identified a novel homozygous deletion in BTG4 (c.580_616del) in the affected individual. The deletion results in frameshift and replacement of the last 29 residues (aa195-223) with 66 random amino acids. The mutated amino acid residues are highly conserved among mammalian species. Co-immunoprecipitation in 293 T cells showed that the mutation abolished the interaction between BTG4 and PABPN1L. CONCLUSION: This study conforms previous studies and expands the mutational spectrum of BTG4. Our findings prove the functional importance of the C-terminal of BTG4. BTG4 is a potential diagnostic and therapeutic target for patients suffering from zygotic cleavage failure.
Subject(s)
Infertility, Female , Animals , Female , Humans , Cell Cycle Proteins/genetics , Fertilization in Vitro/methods , Homozygote , Infertility, Female/genetics , Infertility, Female/pathology , Mammals , Mutation/genetics , Poly(A)-Binding Proteins/genetics , Sequence DeletionABSTRACT
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.
Subject(s)
Dengue , Hyperglycemia , Animals , Mice , Protein Biosynthesis , Phosphatidylinositol 3-Kinases/metabolism , RNA, Messenger/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Viral Proteins/metabolism , Hyperglycemia/complicationsABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factor-4G , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Poly(A)-Binding Proteins/genetics , Polyribosomes/metabolism , Protein Binding , Protein Biosynthesis , RNA, Messenger/metabolism , RibonucleoproteinsABSTRACT
Dual leucine zipper kinase (DLK) plays a pivotal role in the development, degeneration, and regeneration of neurons. DLK can regulate gene expression post-transcriptionally, but the underlying mechanism remains poorly understood. The Drosophila DLK, Wallenda (Wnd), regulates the expression of Down syndrome cell adhesion molecule (Dscam) to control presynaptic arbor growth. This regulation is mediated by the 3' untranslated region (3'UTR) of Dscam mRNA, which suggests that RNA binding proteins (RBPs) mediate DLK function. We performed a genome-wide cell-based RNAi screen of RBPs and identified the cytoplasmic poly(A)-binding protein, pAbp, as an RBP that mediates Wnd-induced increase in Dscam expression. Genetic analysis shows that Wnd requires pAbp for promoting presynaptic arbor growth and for enhancing Dscam expression. Our analysis revealed that Dscam mRNAs harbor short poly(A) tails. We identified a region in Dscam 3'UTR that specifically interacts with pAbp. Removing this region significantly reduced Wnd-induced increase in Dscam expression. These suggest that a noncanonical interaction of PABP with the 3'UTR of target transcripts is essential for DLK functions.SIGNIFICANCE STATEMENT The kinase DLK plays key roles in a multitude of neuronal responses, including axon development, neurodegeneration, and nerve injury. Previous studies show that DLK acts via mRNAs to regulate protein synthesis, but how DLK does so is poorly understood. This study demonstrates that DLK regulates the synthesis of Dscam through the poly(A)-binding protein PABP-C. Whereas PABP-C is known as a general translational activator, our study shows that DLK-mediated Dscam expression involves a noncanonical interaction between PABP-C and the Dscam mRNA, which leads to a selective regulation of Dscam translation by PABP-C. Thus, our study provides novel insights into the mechanisms that underlie the function of DLK and regulation of gene expression of PABP-C.
Subject(s)
Drosophila , Leucine Zippers , 3' Untranslated Regions/genetics , Animals , Drosophila/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/geneticsABSTRACT
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.