RESUMO
PURPOSE: The study aimed to investigate the role of PABPC1 in developmental delay (DD). METHODS: Children were examined by geneticists and pediatricians. Variants were identified using exome sequencing and standard downstream bioinformatics pipelines. We performed in silico molecular modeling and coimmunoprecipitation to test if the variants affect the interaction between PABPC1 and PAIP2. We performed in utero electroporation of mouse embryo brains to enlighten the function of PABPC1. RESULTS: We describe 4 probands with an overlapping phenotype of DD, expressive speech delay, and autistic features and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms were seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to messenger RNA metabolism-related proteins, such as PAIP2. Coimmunoprecipitation confirmed this because it showed a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. Wild-type Pabpc1 could rescue this disturbance, whereas 3 of the 4 variants did not. CONCLUSION: Pathogenic variants in the PABP domain lead to DD, possibly because of interference with the translation initiation and subsequently an impaired neurogenesis in cortical development.
Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Proteína I de Ligação a Poli(A)/metabolismo , Animais , Criança , Deficiências do Desenvolvimento/genética , Heterozigoto , Humanos , Deficiência Intelectual/genética , Camundongos , Transtornos do Neurodesenvolvimento/genética , Proteína I de Ligação a Poli(A)/química , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Sequenciamento do ExomaRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.
Assuntos
Atrofia Muscular/patologia , Distrofia Muscular Oculofaríngea/patologia , Proteína I de Ligação a Poli(A)/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Testes Genéticos , Humanos , Leupeptinas/farmacologia , Leupeptinas/uso terapêutico , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/tratamento farmacológico , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutação , Proteína I de Ligação a Poli(A)/química , Estudo de Prova de Conceito , Agregados Proteicos/efeitos dos fármacosRESUMO
Infertility affects 10-15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A recent study showed that poly(A)binding protein nuclear 1-like (PABPN1L) recruited BTG anti-proliferation factor 4 (BTG4) to mRNA 3'-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated a PABPN1L-antibody and found "ring-like" PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed "ring-like" aggregates in vitro. This phenomenon is similar with CCR4-NOT catalytic subunit, CCR4-NOT transcription complex subunit 7 (CNOT7), when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l-/- and Pabpn1l tm1a/tm1a) simulating the intron 1-exon 2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l-/- MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-â/+âzygote. Both the Btg4 and CCR4-NOT transcription complex subunit 6 like (Cnot6l) genes are necessary for the deadenylation process and Pabpn1l-/- resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-â/+â zygote and 84.2% genes stabilized in the Cnot6l-â/+âzygote were also stabilized in Pabpn1l-â/+â zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4-NOT-mediated maternal mRNA degradation and PABPN1L variants on intron 1-exon 2 could be a genetic marker of female infertility.
Assuntos
Citoplasma/química , Oócitos/ultraestrutura , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/fisiologia , Agregados Proteicos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/química , Humanos , Infertilidade Feminina , Masculino , Camundongos , Camundongos Knockout , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , RNA Mensageiro/metabolismo , Receptores CCR4/genética , Receptores CCR4/fisiologia , Zigoto/metabolismoRESUMO
Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.
Assuntos
Proteína I de Ligação a Poli(A)/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ribonucleoproteínas/química , Motivos de Aminoácidos , Células Cultivadas , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Domínios ProteicosRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by ptosis, dysphagia, and weakness of proximal limbs. OPMD is caused by the expansion of polyalanine in poly(A)-binding protein, nuclear 1 (PABPN1). Although mitochondrial abnormality has been proposed as the possible etiology, the molecular pathogenesis is still poorly understood. The aim of the study was to specify the mechanism by which expanded PABPN1 causes mitochondrial dysfunction in OPMD. We evaluated whether transgenic mouse model of OPMD, by expressing expanded PABPN1, indeed causes mitochondrial abnormality associated with muscle degeneration. We also investigated the mechanism by which expanded PABPN1 would cause mitochondrial dysfunction in the mouse and cell models of OPMD. Mitochondrial localization of PABPN1 was observed in the muscle fibers of patients with OPMD. Moreover, abnormal accumulation of PABPN1 on the inner membrane of mitochondria and reduced expression of OXPHOS complexes were detected in the muscle fibers of the transgenic mice expressing expanded human PABPN1 with a 13-alanine stretch. In cells expressing PABPN1 with a 10-alanine or 18-alanine stretch, both types of PABPN1 accumulated in the mitochondria and interacted with TIM23 mitochondrial protein import complex, but PABPN1 with 18-alanine stretch decreased the cell viability and aggresome formation. We proposed that the abnormal accumulation of expanded PABPN1 in mitochondria may be associated with mitochondrial abnormality in OPMD.
Assuntos
Mitocôndrias Musculares/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Estudos de Casos e Controles , Sobrevivência Celular , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias Musculares/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Oculofaríngea/patologia , Proteínas Mutantes/química , Fosforilação Oxidativa , Proteína I de Ligação a Poli(A)/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Fragile X mental retardation protein (FMRP), a key determinant of normal brain development and neuronal plasticity, plays critical roles in nucleocytoplasmic shuttling of mRNAs. However, the factors involved in FMRP nuclear localization remain to be determined. Using cross-species sequence comparison, we show that an aspartate in position 132 (D132), located within the conserved nuclear localization signal (NLS) of FMRP, appears in human and other mammals, while glutamate 132 (E132) appears in rodents and birds. Human FMRP-D132E alters the secondary structure of the protein and reduces its nuclear localization, while the reciprocal substitution in mouse FMRP-E132D promotes its nuclear localization. Human FMRP could interact with poly(A)-binding protein 1 (PABP1) which is impeded by the D132E mutation. Reversely, mouse FMRP could not interact with PABP1, but the E132D mutation leads to the FMRP-PABP1 interaction. We further show that overexpression of human FMRP-D132E mutant promotes the formation of cytoplasmic aggregates of PABP1 in human cells, but not of mouse FMRP-E132D in mouse cells. PABP1 knockdown reduces the nuclear localization of human FMRP, but not mouse FMRP. Furthermore, RNase A treatment decreases the PABP1 levels in the anti-V5-immunoprecipitates using the V5-hFMRP-transfected cells, suggesting an interaction between human FMRP and PABP1 in an RNA-dependent fashion. Thus, our data suggest that the FMRP protein with the human-used D132 accommodates a novel protein-RNA-protein interaction which may implicate a connection between FMRP residue transition and neural evolution.
Assuntos
Núcleo Celular/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , RNA/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proteína do X Frágil da Deficiência Intelectual/química , Proteína do X Frágil da Deficiência Intelectual/genética , Células HEK293 , Humanos , Camundongos , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , RNA/química , RNA/genética , Especificidade da EspécieRESUMO
Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Leishmania/genética , Proteína I de Ligação a Poli(A)/química , Trans-Splicing/genética , Cristalografia por Raios X , Fator de Iniciação 4E em Eucariotos/genética , Ligantes , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação ao Cap de RNA/química , Proteínas de Ligação ao Cap de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
Assuntos
Proteína II de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Trypanosoma brucei brucei/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Ligação Proteica , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMO
Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.
Assuntos
Apoptose , Exorribonucleases/metabolismo , Mitocôndrias/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Citocromos c/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Células HCT116 , Humanos , Membranas Mitocondriais/metabolismo , Conformação de Ácido Nucleico , Permeabilidade , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/química , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Translational regulation plays a central role during post-meiotic development of male germ cells. Previous studies suggested that P-element induced wimpy testis like 1 (PIWIL1), a PIWI-interacting RNA (piRNA) binding protein that is critical for sperm development, participates in the maintenance and translational regulation of post-meiotic mRNAs in haploid spermatids. However, how PIWIL1 regulates protein translation remains largely unclear. Using biochemical assays, we show here that PIWIL1 utilizes different domains to interact with post-meiotic mRNAs and Poly-A binding protein cytoplasmic 1 (PABPC1), a general protein translation regulator. PIWIL1 binds 3'-untranslated regions (3'-UTRs) of several spermiogenic mRNAs via its N-terminal domain, whereas its interactions with PABPC1 are mediated through its N- and C-terminal domains in an RNA-dependent manner. Using a heterologous cell system, we analyzed its effects on protein translation via luciferase reporter assay and sucrose gradient sedimentation. It was found that PIWIL1 augments protein translation with PABPC1 in the presence of 3'-UTRs of post-meiotic mRNAs. While both the N-terminal RNA recognition motif (RRM) domain and the central linker region of PABPC1 stimulate translation, only the PIWI Argonaute and Zwille (PAZ) domain of PIWIL1 positively affects translation of reporter mRNAs. Interestingly, the PAZ domain was found absent from polysomal fractions, in contrast to the N- and C-terminal domains of PIWIL1. Taken together, the results suggest that PIWIL1 interacts with various partners using different domains and participates in translational regulation partly through 3'-UTRs. It will be of interest to further explore how PIWIL1 elicit its versatile functions, including translational regulation of post-meiotic mRNAs through intrinsic structural changes and extrinsic signals during mouse spermiogenesis under more physiological settings.
Assuntos
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Células HEK293 , Humanos , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína I de Ligação a Poli(A)/genética , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testículo/metabolismoRESUMO
Eukaryotic mature mRNAs possess a poly adenylate tail (poly(A)), to which multiple molecules of poly(A)-binding protein C1 (PABPC1) bind. PABPC1 regulates translation and mRNA metabolism by binding to regulatory proteins. To understand functional mechanism of the regulatory proteins, it is necessary to reveal how multiple molecules of PABPC1 exist on poly(A). Here, we characterize the structure of the multiple molecules of PABPC1 on poly(A), by using transmission electron microscopy (TEM), chemical cross-linking, and NMR spectroscopy. The TEM images and chemical cross-linking results indicate that multiple PABPC1 molecules form a wormlike structure in the PABPC1-poly(A) complex, in which the PABPC1 molecules are linearly arrayed. NMR and cross-linking analyses indicate that PABPC1 forms a multimer by binding to the neighbouring PABPC1 molecules via interactions between the RNA recognition motif (RRM) 2 in one molecule and the middle portion of the linker region of another molecule. A PABPC1 mutant lacking the interaction site in the linker, which possesses an impaired ability to form the multimer, reduced the in vitro translation activity, suggesting the importance of PABPC1 multimer formation in the translation process. We therefore propose a model of the PABPC1 multimer that provides clues to comprehensively understand the regulation mechanism of mRNA translation.
Assuntos
Poli A/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Mutação , Proteína I de Ligação a Poli(A)/genética , Ligação Proteica , Multimerização Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
Increasing evidence indicates that alternative processing of mRNA, including alternative splicing, 3' alternative polyadenylation, and regulation of mRNA stability/translation, represents a major mechanism contributing to protein diversification. For example, in alternative polyadenylation, the 3' end of the immunoglobulin heavy chain mRNA is processed during B cell differentiation, and this processing involves RNA-binding proteins. hnRNPLL (heterogeneous nuclear ribonucleoprotein L-like protein) is an RNA-binding protein expressed in terminally differentiated lymphocytes, such as memory T cells and plasma cells. hnRNPLL regulates various processes of RNA metabolism, including alternative pre-mRNA splicing and RNA stability. In plasma cells, hnRNPLL also regulates the transition from the membrane isoform of the immunoglobulin heavy-chain (mIgH) to the secreted isoform (sIgH), but the precise mechanism remains to be identified. In this study, we report that hnRNPLL specifically associates with cytoplasmic PABPC1 (poly(A)-binding protein 1) in both T cells and plasma cells. We found that although PABPC1 is not required for the alternative splicing of CD45, a primary target of hnRNPLL in lymphocytes, PABPC1 does promote the binding of hnRNPLL to the immunoglobulin mRNA and regulates switching from mIgH to sIgH in plasma cells. Given the recently identified role of PABPC1 in mRNA alternative polyadenylation, our findings suggest that PABPC1 recruits hnRNPLL to the 3'-end of RNA and regulates the transition from membrane Ig to secreted Ig through mRNA alternative polyadenylation. In conclusion, our study has revealed a mechanism that regulates immunoglobulin secretion in B cells via cooperation between a plasma cell-specific RBP (hnRNPLL) and a universally expressed RBP (PABPC1).
Assuntos
Citoplasma/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Plasmócitos/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Poliadenilação , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Imunoprecipitação , Células Jurkat , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/citologia , Plasmócitos/imunologia , Proteína I de Ligação a Poli(A)/antagonistas & inibidores , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Baço/citologia , Baço/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Coactivator associated arginine methyltransferase 1 (CARM1) is a member of the protein arginine methyltransferase (PRMT) family and methylates a range of proteins in eukaryotic cells. Overexpression of CARM1 is implicated in a number of cancers, and it is therefore seen as a potential therapeutic target. Peptide sequences derived from the well-defined CARM1 substrate poly(A)-binding protein 1 (PABP1) were covalently linked to an adenosine moiety as in the AdoMet cofactor to generate transition state mimics. These constructs were found to be potent CARM1 inhibitors and also formed stable complexes with the enzyme. High-resolution crystal structures of CARM1 in complex with these compounds confirm a mode of binding that is indeed reflective of the transition state at the CARM1 active site. Given the transient nature of PRMT-substrate complexes, such transition state mimics represent valuable chemical tools for structural studies aimed at deciphering the regulation of arginine methylation mediated by the family of arginine methyltransferases.
Assuntos
Peptídeos/farmacologia , Proteína I de Ligação a Poli(A)/química , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Adenosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidoresRESUMO
The disease oculopharyngeal muscular dystrophy is caused by alanine codon trinucleotide expansions in the N-terminal segment of the nuclear poly(A) binding protein PABPN1. As histochemical features of the disease, intranuclear inclusions of PABPN1 have been reported. Whereas the purified N-terminal domain of PABPN1 forms fibrils in an alanine-dependent way, fibril formation of the full-length protein occurs also in the absence of alanines. Here, we addressed the question whether the stability of the RNP domain or domain swapping within the RNP domain may add to fibril formation. A variant of full-length PABPN1 with a stabilizing disulfide bond at position 185/201 in the RNP domain fibrillized in a redox-sensitive manner suggesting that the integrity of the RNP domain may contribute to fibril formation. Thermodynamic analysis of the isolated wild-type and the disulfide-linked RNP domain showed two state unfolding/refolding characteristics without detectable intermediates. Quantification of the thermodynamic stability of the mutant RNP domain pointed to an inverse correlation between fibril formation of full-length PABPN1 and the stability of the RNP domain.
Assuntos
Amiloide/química , Amiloide/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Cinética , Conformação Proteica , Estrutura Terciária de Proteína , TemperaturaRESUMO
Oculopharyngeal muscular dystrophy (OPMD), a polyalanine myopathy, occurs due to expansion of homo-polyalanine stretch in normal polyadenylating binding protein nuclear 1 (PABPN1) protein from Ala10 to Ala11-17. Therefore, the conformational behavior of polyalanine peptides with n = 10-17, with and without terminal protecting groups, have been investigated with different starting geometries in water by molecular dynamics simulation studies. Alanine peptides are shown to give rise to unordered structure irrespective of starting geometry and not more than two residues at a stretch have the same/similar set of φ, ψ values. However, the final structure with terminal protecting groups look like ß-strand. Unprotected poly-Ala peptides adopt twisted ß-hairpin/multi hairpin like structure with increasing chain length. The number of residues having φ, ψ values in collagen region is found to be less in peptides with unprotected termini as compared to peptides with protected termini of same chain length. The results have been supported by recent synchrotron radiation circular dichroism spectroscopy of polyproline II and unordered secondary structures. Opening of the helical structure in poly-Ala peptides with protecting groups has been shown to take place from C-terminal and in peptides without protecting groups opening of helix starts from both terminals. Further, opening of helix takes more time in poly-Ala peptides without terminal protecting groups. The deviations in amide bond planarity have been discussed and compared with available experimental and computational results.
Assuntos
Distrofia Muscular Oculofaríngea/genética , Peptídeos/química , Proteína I de Ligação a Poli(A)/química , Amidas/química , Humanos , Simulação de Dinâmica Molecular , Distrofia Muscular Oculofaríngea/patologia , Peptídeos/genética , Proteína I de Ligação a Poli(A)/genética , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.
Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , Regulação da Expressão Gênica , Degradação do RNAm Mediada por Códon sem Sentido , Códon sem Sentido/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação Eucariótico 4G/química , Humanos , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Proto-Oncogênicas c-ets/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The cytoplasmic poly(A) binding protein 1 (PABPC1) is an essential eukaryotic translational initiation factor first described over 40 years ago. Most studies of PABPC1 have focused on its N-terminal RRM domains, which bind the mRNA 3' poly(A) tail and 5' translation complex eIF4F via eIF4G; however, the protein also contains a C-terminal MLLE domain that binds a peptide motif, termed PAM2, found in many proteins involved in translation regulation and mRNA metabolism. Studies over the past decade have revealed additional functions of PAM2-containing proteins (PACs) in neurodegenerative diseases, circadian rhythms, innate defense, and ubiquitin-mediated protein degradation. Here, we summarize functional and structural studies of the MLLE/PAM2 interaction and discuss the diverse roles of PACs.
Assuntos
Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/classificação , RNA Mensageiro/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/químicaRESUMO
The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of â¼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.
Assuntos
Motivos de Aminoácidos , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Técnicas de Inativação de Genes , Variação Genética , Mutação , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA , Relação Estrutura-AtividadeRESUMO
RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.
Assuntos
Biotina/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/química , Mapeamento de Interação de Proteínas , Proteoma/análise , Sequência de Aminoácidos , Bioensaio , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas ELAV/química , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 1 , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Mapas de Interação de Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estreptavidina/químicaRESUMO
The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.