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1.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804860

RESUMO

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.


Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcriptoma
2.
Nat Commun ; 12(1): 1988, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790291

RESUMO

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Ácidos Siálicos/metabolismo , Regulação Alostérica , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Motivos de Nucleotídeos/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Repressoras/genética
3.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809732

RESUMO

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.


Assuntos
Paenibacillus polymyxa/metabolismo , Riboswitch/genética , Serina/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Sequência Conservada , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Nitrogenase/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Paenibacillus polymyxa/efeitos dos fármacos , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/crescimento & desenvolvimento , RNA Bacteriano/química , RNA Bacteriano/genética , Serina/farmacologia
4.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810132

RESUMO

Riboswitches are segments of noncoding RNA that bind with metabolites, resulting in a change in gene expression. To understand the molecular mechanism of gene regulation in a fluoride riboswitch, a base-pair opening dynamics study was performed with and without ligands using the Bacillus cereus fluoride riboswitch. We demonstrate that the structural stability of the fluoride riboswitch is caused by two steps depending on ligands. Upon binding of a magnesium ion, significant changes in a conformation of the riboswitch occur, resulting in the greatest increase in their stability and changes in dynamics by a fluoride ion. Examining hydrogen exchange dynamics through NMR spectroscopy, we reveal that the stabilization of the U45·A37 base-pair due to the binding of the fluoride ion, by changing the dynamics while maintaining the structure, results in transcription regulation. Our results demonstrate that the opening dynamics and stabilities of a fluoride riboswitch in different ion states are essential for the genetic switching mechanism.


Assuntos
Bacillus cereus/genética , Pareamento de Bases , Fluoretos/química , Genes Bacterianos , Riboswitch , Aptâmeros de Nucleotídeos , Sequência de Bases , Catálise , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos
5.
Methods Mol Biol ; 2278: 225-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33649960

RESUMO

In this chapter, we present a generic method to achieve de novo genome assembly and methylome analysis of Bifidobacterium genomes using the Pacbio SMRT sequencing and SMRT Link pipeline. The methods described here cover the de novo Pacbio or hybrid Illumina-Pacbio assembly ideally intended to obtain a complete genome sequence, followed by the detection of base modifications and methylome analysis.The identified DNA motifs obtained by methylome analysis can be used to predict active restriction-modification (RM) systems in Bifidobacterium strains. The presence of active RM systems and knowledge on their target motifs may guide selection of suitable cloning or mutagenesis vectors for bifidobacteria.


Assuntos
Bifidobacterium/genética , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Metilação de DNA , DNA Bacteriano/genética , Motivos de Nucleotídeos , Software
6.
Nat Commun ; 12(1): 1384, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654077

RESUMO

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Edição de Genes , Células HEK293 , Humanos , Motivos de Nucleotídeos/genética
7.
Nucleic Acids Res ; 49(5): 2488-2508, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33533919

RESUMO

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.


Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Epigênese Genética , Antígeno 2 Relacionado a Fos/metabolismo , Humanos , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fator de Transcrição AP-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
8.
Nucleic Acids Res ; 49(5): 2946-2958, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33577684

RESUMO

RBM45 is an RNA-binding protein involved in neural development, whose aggregation is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). However, the mechanisms of RNA-binding and aggregation of RBM45 remain unelucidated. Here, we report the crystal structure of the N-terminal tandem RRM domains of human RBM45 in complex with single-stranded DNA (ssDNA). Our structural and biochemical results revealed that both the RRM1 and RRM2 of RBM45 recognized the GAC sequence of RNA/ssDNA. Two aromatic residues and an arginine residue in each RRM were critical for RNA-binding, and the interdomain linker was also involved in RNA-binding. Two RRMs formed a pair of antiparallel RNA-binding sites, indicating that the N-terminal tandem RRM domains of RBM45 bound separate GAC motifs in one RNA strand or GAC motifs in different RNA strands. Our findings will be helpful in the identification of physiologic targets of RBM45 and provide evidence for understanding the physiologic and pathologic functions of RBM45.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas de Ligação a RNA/química , RNA/química , Cristalografia por Raios X , DNA de Cadeia Simples/química , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , RNA/metabolismo , Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/metabolismo
9.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562358

RESUMO

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the "Find Individual Motif Occurrences" (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.


Assuntos
Adenosina Trifosfatases/genética , Movimento Celular , Proteínas Cromossômicas não Histona/genética , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Neovascularização Patológica/patologia , RNA Circular/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Motivos de Nucleotídeos , Prognóstico , RNA Circular/genética , Fatores de Processamento de Serina-Arginina/genética , Células Tumorais Cultivadas
10.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567767

RESUMO

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor and plays a key role in tumor progression. The C-rich DNA sequence of VEGF promoter can form i-motif structure, which is a potential target for the development of novel anticancer agents. However, there is a limited number of chemotypes as the selective ligands of VEGF promoter i-motif, which leaves much room for development. Herein, we report the discovery of the natural oleanolic acid scaffold as a novel chemotype for the development of selective ligands of VEGF i-motif. A series of oleanolic acid derivatives as VEGF promoter i-motif ligands were synthesized. Subsequent evaluations showed that 3c could selectively bind to and stabilize VEGF promoter i-motif without significant binding to G-quadruplex, duplex DNA, and other oncogene i-motifs. Cell-based assays indicated that 3c could effectively downregulate VEGF gene transcription and expression in MCF-7 cells, inhibit tumor cells proliferation and migration, and induce cancer cells apoptosis. This work provides evidence of VEGF promoter i-motif as an anticancer target and will facilitate future efforts for the discovery of oleanolic acid-based selective ligands of VEGF promoter i-motif.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Motivos de Nucleotídeos , Ácido Oleanólico/farmacologia , Regiões Promotoras Genéticas , Fator A de Crescimento do Endotélio Vascular/genética , Antineoplásicos/química , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Feminino , Humanos , Conformação de Ácido Nucleico , Ácido Oleanólico/química , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/química
11.
Nucleic Acids Res ; 49(4): 2317-2332, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33524154

RESUMO

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).


Assuntos
DNA Circular/química , Pareamento de Bases , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Saccharomyces cerevisiae/genética , Estereoisomerismo , Telômero/química
12.
Nucleic Acids Res ; 49(4): 2065-2084, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33555350

RESUMO

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.


Assuntos
Replicação do DNA , DNA/química , DNA/metabolismo , Quadruplex G , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutação , Motivos de Nucleotídeos , Nucleotidiltransferases/genética , Ligação Proteica
13.
Nat Genet ; 53(3): 354-366, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33603233

RESUMO

The arrangement (syntax) of transcription factor (TF) binding motifs is an important part of the cis-regulatory code, yet remains elusive. We introduce a deep learning model, BPNet, that uses DNA sequence to predict base-resolution chromatin immunoprecipitation (ChIP)-nexus binding profiles of pluripotency TFs. We develop interpretation tools to learn predictive motif representations and identify soft syntax rules for cooperative TF binding interactions. Strikingly, Nanog preferentially binds with helical periodicity, and TFs often cooperate in a directional manner, which we validate using clustered regularly interspaced short palindromic repeat (CRISPR)-induced point mutations. Our model represents a powerful general approach to uncover the motifs and syntax of cis-regulatory sequences in genomics data.


Assuntos
Biologia Computacional/métodos , Motivos de Nucleotídeos , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Aprendizado Profundo , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Proteína Homeobox Nanog/metabolismo , Redes Neurais de Computação , Fator 3 de Transcrição de Octâmero/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição SOXB1/metabolismo
14.
Nat Protoc ; 16(3): 1511-1547, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33547443

RESUMO

The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Humanos , Motivos de Nucleotídeos/genética , RNA Guia/genética , Projetos de Pesquisa
15.
Nat Struct Mol Biol ; 28(1): 103-117, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398172

RESUMO

Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.


Assuntos
Regulação da Expressão Gênica/genética , Complexo Repressor Polycomb 2/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Polimerase II/metabolismo , RNA/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA/genética , Transcrição Genética/genética , Ativação Transcricional/genética
16.
Nucleic Acids Res ; 49(3): 1708-1723, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33450012

RESUMO

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.


Assuntos
Enzimas de Restrição do DNA/química , DNA/química , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Domínio Catalítico , Cristalografia por Raios X , DNA de Cadeia Simples/química , Modelos Moleculares , Motivos de Nucleotídeos , Domínios Proteicos , Espalhamento a Baixo Ângulo , Vibrio/enzimologia , Difração de Raios X
17.
Nucleic Acids Res ; 49(3): 1724-1736, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33503265

RESUMO

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.


Assuntos
DNA/química , Quadruplex G , Cristalografia por Raios X , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Motivos de Nucleotídeos , Açúcares/química
18.
Nat Commun ; 12(1): 328, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436566

RESUMO

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Bacteriano , RNA de Transferência/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilação , Anticódon/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Hidrólise , Metilação , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo
19.
Nat Commun ; 12(1): 33, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397927

RESUMO

The Origin Recognition Complex (ORC) is an evolutionarily conserved six-subunit protein complex that binds specific sites at many locations to coordinately replicate the entire eukaryote genome. Though highly conserved in structure, ORC's selectivity for replication origins has diverged tremendously between yeasts and humans to adapt to vastly different life cycles. In this work, we demonstrate that the selectivity determinant of ORC for DNA binding lies in a 19-amino acid insertion helix in the Orc4 subunit, which is present in yeast but absent in human. Removal of this motif from Orc4 transforms the yeast ORC, which selects origins based on base-specific binding at defined locations, into one whose selectivity is dictated by chromatin landscape and afforded with plasticity, as reported for human. Notably, the altered yeast ORC has acquired an affinity for regions near transcriptional start sites (TSSs), which the human ORC also favors.


Assuntos
Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Fúngico/metabolismo , Fase G2/genética , Genoma Fúngico , Humanos , Modelos Genéticos , Mutação/genética , Nucleossomos/metabolismo , Motivos de Nucleotídeos/genética , Complexo de Reconhecimento de Origem/química , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Processos Estocásticos , Sítio de Iniciação de Transcrição
20.
Development ; 148(1)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441380

RESUMO

The number of long noncoding RNAs (lncRNAs) with characterized developmental and cellular functions continues to increase, but our understanding of the molecular mechanisms underlying lncRNA functions, and how they are dictated by RNA sequences, remains limited. Relatively short, conserved sequence motifs embedded in lncRNA transcripts are often important determinants of lncRNA localization, stability and interactions. Identifying such RNA motifs remains challenging due to the substantial length of lncRNA transcripts and the rapid evolutionary turnover of lncRNA sequences. Nevertheless, the recent discovery of specific RNA elements, together with their experimental interrogation, has enabled the first step in classifying heterogeneous lncRNAs into sub-groups with similar molecular mechanisms and functions. In this Review, we focus on lncRNAs with roles in development, cell differentiation and normal physiology in vertebrates, and we discuss the sequence elements defining their functions. We also summarize progress on the discovery of regulatory RNA sequence elements, as well as their molecular functions and interaction partners.


Assuntos
Diferenciação Celular/genética , Motivos de Nucleotídeos/genética , RNA Longo não Codificante/genética , Animais , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Estabilidade de RNA/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo
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