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1.
Mol Biol (Mosk) ; 53(4): 663-673, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397440

RESUMO

Malignant cutaneous melanoma (CM) is an extremely aggressive cancer characterized by a high level of metastatic activity and unfavorable prognosis due to a high incidence of relapses, as well as resistance to standard chemotherapy. Cutaneous melanoma accounts for 80% of deaths from malignant skin tumors. Nucleolin/C23 and nucleophosmin/B23, which constitute altogether ~70% of the nucleolus volume, are promising targets for molecular therapy of melanoma. These proteins perform many important functions in the cell, so disruption of the NCL and/or NPM gene structure and abnormal expression of the C23 and B23 proteins they encode, can lead to unlimited cell proliferation and progression of a tumor. Therefore, investigation of the structure and expression of these genes is a topical problem, which is important for understanding the mechanisms of CM carcinogenesis and for the development of new therapeutic approaches. This paper describes new NCL and NPM polymorphisms, as well as the levels of C23 and B23 expression in normal tissues, CM and mucosal melanoma.


Assuntos
Melanoma/genética , Melanoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Proliferação de Células , Humanos , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Fosfoproteínas/biossíntese , Fosfoproteínas/química , Polimorfismo Genético , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Neoplasias Cutâneas/tratamento farmacológico
3.
Nature ; 571(7765): 424-428, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292544

RESUMO

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.


Assuntos
Adenosina/análogos & derivados , Compartimento Celular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Transição de Fase , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico
4.
BMC Evol Biol ; 19(1): 149, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337330

RESUMO

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.


Assuntos
Antivirais/metabolismo , Vírus de DNA/genética , Moluscos/virologia , Edição de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Teorema de Bayes , Vírus de DNA/fisiologia , Regulação da Expressão Gênica , Genoma Viral , Modelos Moleculares , Moluscos/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
5.
Protein Pept Lett ; 26(8): 601-619, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215361

RESUMO

The interactions between RNAs and proteins play critical roles in many biological processes. Therefore, characterizing these interactions becomes critical for mechanistic, biomedical, and clinical studies. Many experimental methods can be used to determine RNA-protein interactions in multiple aspects. However, due to the facts that RNA-protein interactions are tissuespecific and condition-specific, as well as these interactions are weak and frequently compete with each other, those experimental techniques can not be made full use of to discover the complete spectrum of RNA-protein interactions. To moderate these issues, continuous efforts have been devoted to developing high quality computational techniques to study the interactions between RNAs and proteins. Many important progresses have been achieved with the application of novel techniques and strategies, such as machine learning techniques. Especially, with the development and application of CLIP techniques, more and more experimental data on RNA-protein interaction under specific biological conditions are available. These CLIP data altogether provide a rich source for developing advanced machine learning predictors. In this review, recent progresses on computational predictors for RNA-protein interaction were summarized in the following aspects: dataset, prediction strategies, and input features. Possible future developments were also discussed at the end of the review.


Assuntos
Aprendizado de Máquina , Proteínas de Ligação a RNA/química , RNA/química , Algoritmos , Biologia Computacional/métodos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
6.
J Chem Theory Comput ; 15(8): 4687-4698, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31251056

RESUMO

Small-angle X-ray and small-angle neutron scattering (SAXS/SANS) provide unique structural information on biomolecules and their complexes in solution. SANS may provide multiple independent data sets by means of contrast variation experiments, that is, by measuring at different D2O concentrations and different perdeuteration conditions of the biomolecular complex. However, even the combined data from multiple SAXS/SANS sets is by far insufficient to define all degrees of freedom of a complex, leading to a significant risk of overfitting when refining biomolecular structures against SAXS/SANS data. Hence, to control against overfitting, the low-information SAXS/SANS data must be complemented by accurate physical models, and, if possible, refined models should be cross-validated against independent data not used during the refinement. We present a method for refining atomic biomolecular structures against multiple sets of SAXS and SANS data using all-atom molecular dynamics simulations. Using the protein citrate synthase and the protein/RNA complex Sxl-Unr-msl2 mRNA as test cases, we demonstrate how multiple SAXS and SANS sets may be used for refinement and cross-validation, thereby excluding overfitting during refinement. For the Sxl-Unr-msl2 complex, we find that perdeuteration of the Unr domain leads to a unique, slightly compacted conformation, whereas other perdeuteration conditions lead to similar solution conformations compared to the nondeuterated state. In line with our previous method for predicting SAXS curves, SANS curves were predicted with explicit-solvent calculations, taking atomic models for both the hydration layer and the excluded solvent into account, thereby avoiding the use of solvent-related fitting parameters and solvent-reduced neutron scattering lengths. We expect the method to be useful for deriving and validating solution structures of biomolecules and soft-matter complexes, and for critically assessing whether multiple SAXS and SANS sets are mutually compatible.


Assuntos
Simulação de Dinâmica Molecular , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Difração de Raios X , Animais , Citrato (si)-Sintase/química , Proteínas de Ligação a DNA/química , Drosophila , Proteínas de Drosophila/química , Difração de Nêutrons/métodos , RNA/química , Proteínas de Ligação a RNA/química , Suínos , Difração de Raios X/métodos
7.
Nucleic Acids Res ; 47(10): 4958-4969, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31162604

RESUMO

RNA molecules are able to bind proteins, DNA and other small or long RNAs using information at primary, secondary or tertiary structure level. Recent techniques that use cross-linking and immunoprecipitation of RNAs can detect these interactions and, if followed by high-throughput sequencing, molecules can be analysed to find recurrent elements shared by interactors, such as sequence and/or structure motifs. Many tools are able to find sequence motifs from lists of target RNAs, while others focus on structure using different approaches to find specific interaction elements. In this work, we make a systematic analysis of RBP-RNA and RNA-RNA datasets to better characterize the interaction landscape with information about multi-motifs on the same RNAs. To achieve this goal, we updated our BEAM algorithm to combine both sequence and structure information to create pairs of patterns that model motifs of interaction. This algorithm was applied to several RNA binding proteins and ncRNAs interactors, confirming already known motifs and discovering new ones. This landscape analysis on interaction variability reflects the diversity of target recognition and underlines that often both primary and secondary structure are involved in molecular recognition.


Assuntos
Motivos de Nucleotídeos , Proteínas de Ligação a RNA/química , RNA/química , Análise de Sequência de RNA/métodos , Algoritmos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Células HEK293 , Células Hep G2 , Humanos , Células K562 , Camundongos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
8.
Emerg Microbes Infect ; 8(1): 895-908, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31223062

RESUMO

The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essential and non-redundant cellular functions, a Prp8 intein inhibitor is a mechanistically novel antifungal agent. In this report, we demonstrated that cisplatin, an FDA-approved cancer drug, significantly arrested growth of Prp8 intein-containing fungi C. neoformans and C. gattii, but only poorly inhibited growth of intein-free Candida species. These results suggest that cisplatin arrests fungal growth through specific inhibition of the Prp8 intein. Cisplatin was also found to significantly inhibit growth of C. neoformans in a mouse model. Our results further showed that cisplatin inhibited Prp8 intein splicing in vitro in a dose-dependent manner by direct binding to the Prp8 intein. Crystal structures of the apo- and cisplatin-bound Prp8 inteins revealed that two degenerate cisplatin molecules bind at the intein active site. Mutation of the splicing-site residues led to loss of cisplatin binding, as well as impairment of intein splicing. Finally, we found that overexpression of the Prp8 intein in cryptococcal species conferred cisplatin resistance. Overall, these results indicate that the Prp8 intein is a novel antifungal target worth further investigation.


Assuntos
Antifúngicos/administração & dosagem , Cisplatino/administração & dosagem , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/genética , Inteínas , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Antifúngicos/química , Cisplatino/química , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/metabolismo , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência
9.
Nat Commun ; 10(1): 2593, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197139

RESUMO

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.


Assuntos
Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Microscopia de Fluorescência , Modelos Animais , Príons/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
10.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
11.
Nat Commun ; 10(1): 1960, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036859

RESUMO

Lin28-dependent oligo-uridylylation of precursor let-7 (pre-let-7) by terminal uridylyltransferase 4/7 (TUT4/7) represses let-7 expression by blocking Dicer processing, and regulates cell differentiation and proliferation. The interaction between the Lin28:pre-let-7 complex and the N-terminal Lin28-interacting module (LIM) of TUT4/7 is required for pre-let-7 oligo-uridylylation by the C-terminal catalytic module (CM) of TUT4/7. Here, we report crystallographic and biochemical analyses of the LIM of human TUT4. The LIM consists of the N-terminal Cys2His2-type zinc finger (ZF) and the non-catalytic nucleotidyltransferase domain (nc-NTD). The ZF of LIM adopts a distinct structural domain, and its structure is homologous to those of double-stranded RNA binding zinc fingers. The interaction between the ZF and pre-let-7 stabilizes the Lin28:pre-let-7:TUT4 ternary complex, and enhances the oligo-uridylylation reaction by the CM. Thus, the ZF in LIM and the zinc-knuckle in the CM, which interacts with the oligo-uridylylated tail, together facilitate Lin28-dependent pre-let-7 oligo-uridylylation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Humanos , MicroRNAs/genética , Ligação Proteica , RNA Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/genética
12.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075852

RESUMO

Lin-28 is an RNA-binding protein that is known for its role in promoting the pluripotency of stem cells. In the present study, Arabian camel Lin-28 (cLin-28) cDNA was identified and analyzed. Full length cLin-28 mRNA was obtained using the reverse transcription polymerase chain reaction (RT-PCR). It was shown to be 715 bp in length, and the open reading frame (ORF) encoded 205 amino acids. The molecular weight and theoretical isoelectric point (pI) of the cLin-28 protein were predicted to be 22.389 kDa and 8.50, respectively. Results from the bioinformatics analysis revealed that cLin-28 has two main domains: an N-terminal cold-shock domain (CSD) and a C-terminal pair of retroviral-type Cysteine3Histidine (CCHC) zinc fingers. Sequence similarity and phylogenetic analysis showed that the cLin-28 protein is grouped together Camelus bactrianus and Bos taurus. Quantitative real-time PCR (qPCR) analysis showed that cLin-28 mRNA is highly expressed in the lung, heart, liver, and esophageal tissues. Peptide mass fingerprint-mass spectrometry (PMF-MS) analysis of the purified cLin-28 protein confirmed the identity of this protein. Comparing the modeled 3D structure of cLin-28 protein with the available protein 3D structure of the human Lin-28 protein confirmed the presence of CSD and retroviral-type CCHC zinc fingers, and high similarities were noted between the two structures by using super secondary structure prediction.


Assuntos
Camelus/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Regulação da Expressão Gênica , Modelos Moleculares , Peptídeos/química , Filogenia , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo
13.
Molecules ; 24(10)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121831

RESUMO

Aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)-derived peptide (AdP) has been developed as a cosmeceutical ingredient for skin anti-aging given its fibroblast-activating (FA) and melanocyte-inhibiting (MI) functions. However, a suitable strategy for the topical delivery of AdP was required due to its low-permeable properties. In this study, FA and MI domains of AdP (FA-AdP and MI-AdP, respectively) were determined by functional domain mapping, where the activities of several fragments of AdP on fibroblast and melanocyte were tested, and a hydrosol-based topical delivery system for these AdP fragments was prepared. The excipient composition of the hydrosol was optimized to maximize the viscosity and drying rate by using Box-Behnken design. The artificial skin deposition of FA-AdP-loaded hydrosol was evaluated using Keshary-Chien diffusion cells equipped with Strat-M membrane (STM). The quantification of the fluorescent dye-tagged FA-AdP in STM was carried out by near-infrared fluorescence imaging. The optimized hydrosol showed 127-fold higher peptide deposition in STM than free FA-AdP (p < 0.05). This work suggests that FA- and MI-AdP are active-domains for anti-wrinkle and whitening activities, respectively, and the hydrosol could be used as a promising cosmetic formulation for the delivery of AdPs to the skin.


Assuntos
Cosmecêuticos/farmacologia , Citocinas/química , Proteínas de Neoplasias/química , Peptídeos/farmacologia , Proteínas de Ligação a RNA/química , Envelhecimento da Pele/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cosmecêuticos/química , Doxorrubicina , Humanos , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Camundongos , Modelos Biológicos , Imagem Óptica , Peptídeos/química , Viscosidade
14.
RNA ; 25(8): 1020-1037, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31110137

RESUMO

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Processamento de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
15.
Mol Cell ; 74(5): 966-981.e18, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31078383

RESUMO

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.


Assuntos
Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos/genética , Humanos , Cinética , Ligação Proteica/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Ribossomos/química , Ribossomos/genética
16.
Nat Commun ; 10(1): 2266, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118463

RESUMO

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.


Assuntos
Modelos Moleculares , RNA Mensageiro/metabolismo , Motivos de Ligação ao RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica/fisiologia , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Técnica de Seleção de Aptâmeros , Análise de Sequência de DNA/métodos
17.
Nucleic Acids Res ; 47(7): 3739-3751, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993347

RESUMO

Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.


Assuntos
Endorribonucleases/química , HIV-1/química , RNA Catalítico/química , Proteínas de Ligação a RNA/química , RNA/química , Sítios de Ligação , Endorribonucleases/genética , Genes de RNAr/genética , HIV-1/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/genética , RNA Catalítico/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Termodinâmica
18.
Gen Comp Endocrinol ; 277: 122-129, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951723

RESUMO

Gonadal soma-derived factor (Gsdf) is a unique TGF-ß factor essential for both ovarian and testicular development in Hd-rR medaka (Oryzias latipes). However, the downstream genes regulated by Gsdf signaling remain unknown. Using a high-throughput proteomic approach, we identified a significant increase in the expression of the RNA-binding protein Igf2bp3 in gsdf-deficient ovaries. We verified this difference in transcription and protein expression against normal gonads using real-time PCR quantification and Western blotting. The genomic structure of igf2bp3 and the syntenic flanking segments are highly conserved across fish and mammals. igf2bp3 expression was correlated with oocyte development, which is consistent with the expression of the igf2bp3 ortholog Vg1-RBP/Vera in Xenopus. In contrast to the normal ovary, cysts of H3K27me3- and Igf2bp3-positive germ cells were dramatically increased in the one-month-old gsdf-deficient ovary, indicating that the gsdf depletion led to a dysregulation of Igf2bp3-mediated oocyte development. Our results provide novel insights into the Gsdf-Igf2bp3 signaling mechanisms that underlie the fundamental process of gametogenesis; these mechanisms may be well conserved across phyla.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Oryzias/genética , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta/deficiência , Sequência de Aminoácidos , Animais , Proliferação de Células , Sequência Conservada , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Histonas/metabolismo , Lisina/metabolismo , Masculino , Oogênese/genética , Ovário/embriologia , Ovário/metabolismo , Filogenia , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo
19.
Cell Biol Int ; 43(7): 749-759, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30958636

RESUMO

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e-GFP-6His did co-sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a KD is comparable to some specific MT-associated proteins. Distinct in vitro interaction of RPL22e-GFP with MTs was also observed by TIRF microscopy. In real-time assay, RPL22e-GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random-walk along MTs with diffusion coefficient 0.03 µ2 /s. Deletion of basic areas of RPL22e did not have an impact on KD , and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT-dependent transport and could ameliorate its transport to the nucleus.


Assuntos
Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Bovinos , Cercopithecus aethiops , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Células Vero
20.
Mol Cell ; 74(4): 701-712.e9, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30948266

RESUMO

Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.


Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Leucemia/genética , Complexos Multiproteicos/genética , Receptores CXCR4/genética , Regiões 3' não Traduzidas/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Proteína 3 com Repetições IAP de Baculovírus/química , Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Proteína Semelhante a ELAV 1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/patologia , Complexos Multiproteicos/química , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
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