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1.
Nature ; 584(7820): 279-285, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760005

RESUMO

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes1. One such modification, 8-oxoguanine2 (o8G), is abundant in RNA3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy4. We find that position-specific o8G modifications are generated in seed regions (positions 2-8) of selective miRNAs, and function to regulate other mRNAs through o8G•A base pairing. o8G is induced predominantly at position 7 of miR-1 (7o8G-miR-1) by treatment with an adrenergic agonist. Introducing 7o8G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o8G-miR-1 function in affected phenotypes; the specific inhibition of 7o8G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o8G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression.


Assuntos
Cardiomegalia/genética , Cardiomegalia/patologia , Inativação Gênica , MicroRNAs/química , MicroRNAs/metabolismo , Animais , Pareamento de Bases , Linhagem Celular , Modelos Animais de Doenças , Guanina/análogos & derivados , Guanina/análise , Guanina/química , Guanina/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Ratos , Transcrição Genética/genética , Transcriptoma/genética
2.
Proc Natl Acad Sci U S A ; 117(31): 18224-18230, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680959

RESUMO

Utilizing an ionic liquid strategy, we report crystal structures of salts of free anionic nucleobases and base pairs previously studied only computationally and in the gas phase. Reaction of tetrabutylammonium ([N4444]+) or tetrabutylphosphonium ([P4444]+) hydroxide with adenine (HAd) and thymine (HThy) led to hydrated salts of deprotonated adenine, [N4444][Ad]·2H2O, and thymine, [P4444][Thy]·2H2O, as well as the double salt cocrystal, [P4444]2[Ad][Thy]·3H2O·2HThy. The cocrystal includes the anionic [Ad-(HThy)] base pair which is a stable formation in the solid state that has previously not even been suggested. It exhibits Watson-Crick connectivity as found in DNA but which is unusual for the free neutral base pairs. The stability of the observed anionic bases and their supramolecular formations and hydrates has also been examined by electronic structure calculations, contributing to more insight into how base pairs can bind when a proton is removed and highlighting mechanisms of stabilization or chemical transformation in the DNA chains.


Assuntos
Adenina/química , Pareamento de Bases , Timina/química , Ânions , Cristalografia , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico
3.
Proc Natl Acad Sci U S A ; 117(28): 16333-16338, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601241

RESUMO

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.


Assuntos
Biossíntese de Proteínas/genética , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Pareamento de Bases , Códon/genética , Códon/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
4.
Nucleic Acids Res ; 48(15): 8796-8807, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652019

RESUMO

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.


Assuntos
Citosina/análogos & derivados , DNA/efeitos dos fármacos , Conformação Molecular/efeitos dos fármacos , Termodinâmica , Pareamento de Bases/genética , Ilhas de CpG/genética , Citosina/química , Citosina/farmacologia , DNA/química , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , Cinética , Espectroscopia de Ressonância Magnética , Temperatura de Transição
5.
Nucleic Acids Res ; 48(15): 8675-8685, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687167

RESUMO

Along with nucleobase pairing, base-base stacking interactions are one of the two main types of strong non-covalent interactions that define the unique secondary and tertiary structure of RNA. In this paper we studied two subfamilies of nucleobase-inserted stacking structures: (i) with any base intercalated between neighboring nucleotide residues (base-intercalated element, BIE, i + 1); (ii) with any base wedged into a hydrophobic cavity formed by heterocyclic bases of two nucleotides which are one nucleotide apart in sequence (base-wedged element, BWE, i + 2). We have exploited the growing database of natively folded RNA structures in Protein Data Bank to analyze the distribution and structural role of these motifs in RNA. We found that these structural elements initially found in yeast tRNAPhe are quite widespread among the tertiary structures of various RNAs. These motifs perform diverse roles in RNA 3D structure formation and its maintenance. They contribute to the folding of RNA bulges and loops and participate in long-range interactions of single-stranded stretches within RNA macromolecules. Furthermore, both base-intercalated and base-wedged motifs participate directly or indirectly in the formation of RNA functional centers, which interact with various ligands, antibiotics and proteins.


Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/ultraestrutura , RNA/ultraestrutura , Antibacterianos/química , Pareamento de Bases/genética , Substâncias Intercalantes/química , Ligantes , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleotídeos/química , Nucleotídeos/genética , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
6.
Nucleic Acids Res ; 48(15): 8302-8319, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32725210

RESUMO

We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar structures. The investigated compounds were either free forms or derivatives of adenine, guanine, hypoxanthine, thymine, uracil and cytosine. We divided our findings into categories including types of pairs, protonation patterns and whether they are formed by free bases or substituted ones. We found base pair types that are exclusive to small molecule crystal structures, some that can be found only in RNA containing crystal structures and many that are native to both environments. With a few exceptions, nucleobase protonation generally followed a standard pattern governed by pKa values. The lengths of hydrogen bonds did not depend on whether the nucleobases forming a base pair were charged or not. The reasons why particular nucleobases formed base pairs in a certain way varied significantly.


Assuntos
Bases de Dados de Proteínas , Ligação de Hidrogênio , Conformação Proteica , Proteínas/genética , Adenina/química , Pareamento de Bases/genética , Cristalografia por Raios X , Citosina/química , Guanina/química , Hipoxantina/química , Estrutura Molecular , Proteínas/química , Proteínas/ultraestrutura , Bibliotecas de Moléculas Pequenas/química , Timina/química , Uracila/química
7.
Nucleic Acids Res ; 48(14): 7690-7699, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652015

RESUMO

RNA-small molecule binding is a key regulatory mechanism which can stabilize 3D structures and activate molecular functions. The discovery of RNA-targeting compounds is thus a current topic of interest for novel therapies. Our work is a first attempt at bringing the scalability and generalization abilities of machine learning methods to the problem of RNA drug discovery, as well as a step towards understanding the interactions which drive binding specificity. Our tool, RNAmigos, builds and encodes a network representation of RNA structures to predict likely ligands for novel binding sites. We subject ligand predictions to virtual screening and show that we are able to place the true ligand in the 71st-73rd percentile in two decoy libraries, showing a significant improvement over several baselines, and a state of the art method. Furthermore, we observe that augmenting structural networks with non-canonical base pairing data is the only representation able to uncover a significant signal, suggesting that such interactions are a necessary source of binding specificity. We also find that pre-training with an auxiliary graph representation learning task significantly boosts performance of ligand prediction. This finding can serve as a general principle for RNA structure-function prediction when data is scarce. RNAmigos shows that RNA binding data contains structural patterns with potential for drug discovery, and provides methodological insights for possible applications to other structure-function learning tasks. The source code, data and a Web server are freely available at http://rnamigos.cs.mcgill.ca.


Assuntos
RNA/química , Software , Pareamento de Bases , Sítios de Ligação , Ligantes , Conformação de Ácido Nucleico
8.
Nucleic Acids Res ; 48(13): e77, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496533

RESUMO

A fast-growing number of non-coding RNA structures have been resolved and deposited in Protein Data Bank (PDB). In contrast to the wide range of global alignment and motif search tools, there is still a lack of local alignment tools. Among all the global alignment tools for RNA 3D structures, STAR3D has become a valuable tool for its unprecedented speed and accuracy. STAR3D compares the 3D structures of RNA molecules using consecutive base-pairs (stacks) as anchors and generates an optimal global alignment. In this article, we developed a local RNA 3D structural alignment tool, named LocalSTAR3D, which was extended from STAR3D and designed to report multiple local alignments between two RNAs. The benchmarking results show that LocalSTAR3D has better accuracy and coverage than other local alignment tools. Furthermore, the utility of this tool has been demonstrated by rediscovering kink-turn motif instances, conserved domains in group II intron RNAs, and the tRNA mimicry of IRES RNAs.


Assuntos
Pareamento de Bases , RNA/química , Alinhamento de Sequência/métodos , Análise de Sequência de RNA/métodos , Software , Bases de Dados de Ácidos Nucleicos , Modelos Moleculares
9.
Nucleic Acids Res ; 48(13): 7545-7556, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520325

RESUMO

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem-loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen-Watson-Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.


Assuntos
Dobramento de RNA , Riboswitch , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Pareamento de Bases , Transferência Ressonante de Energia de Fluorescência , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo
10.
Nucleic Acids Res ; 48(14): 7640-7652, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32558908

RESUMO

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , RNA de Cadeia Dupla/química , Pareamento de Bases , DNA de Cadeia Simples/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química
11.
Nature ; 583(7814): 139-144, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32461691

RESUMO

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.


Assuntos
Pareamento de Bases , MicroRNAs/genética , RNA Mensageiro/genética , Sirtuína 1/genética , Proteínas Argonauta/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Complexo de Inativação Induzido por RNA/metabolismo
12.
Acta Crystallogr C Struct Chem ; 76(Pt 5): 513-523, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32367834

RESUMO

The positional change of nitrogen-7 of the RNA constituent guanosine to the bridgehead position-5 leads to the base-modified nucleoside 5-aza-7-deazaguanosine. Contrary to guanosine, this molecule cannot form Hoogsteen base pairs and the Watson-Crick proton donor site N3-H becomes a proton-acceptor site. This causes changes in nucleobase recognition in nucleic acids and has been used to construct stable `all-purine' DNA and DNA with silver-mediated base pairs. The present work reports the single-crystal X-ray structure of 7-iodo-5-aza-7-deazaguanosine, C10H12IN5O5 (1). The iodinated nucleoside shows an anti conformation at the glycosylic bond and an N conformation (O4'-endo) for the ribose moiety, with an antiperiplanar orientation of the 5'-hydroxy group. Crystal packing is controlled by interactions between nucleobase and sugar moieties. The 7-iodo substituent forms a contact to oxygen-2' of the ribose moiety. Self-pairing of the nucleobases does not take place. A Hirshfeld surface analysis of 1 highlights the contacts of the nucleobase and sugar moiety (O-H...O and N-H...O). The concept of pK-value differences to evaluate base-pair stability was applied to purine-purine base pairing and stable base pairs were predicted for the construction of `all-purine' RNA. Furthermore, the 7-iodo substituent of 1 was functionalized with benzofuran to detect motional constraints by fluorescence spectroscopy.


Assuntos
DNA/química , Guanosina/análogos & derivados , Ácidos Nucleicos/química , Purinas/química , Ribonucleosídeos/química , Prata/química , Pareamento de Bases , Cristalografia por Raios X , Guanosina/química , Conformação Molecular
13.
Nucleic Acids Res ; 48(11): 5907-5925, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383760

RESUMO

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.


Assuntos
Elementos Facilitadores Genéticos , Genoma Humano , Guanina , Conformação de Ácido Nucleico , Pareamento de Bases , Quadruplex G , Rearranjo Gênico , Variação Genética , Genômica , Guanina/análise , Humanos , Saccharomyces cerevisiae/genética , Duplicações Segmentares Genômicas , Deleção de Sequência
14.
RNA ; 26(9): 1118-1130, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32414856

RESUMO

Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic conditions are held together by A+-A+ base-pairing also involving base interactions with the phosphate backbone. Traditional UV-melting studies of parallel poly(A) duplexes have typically examined homo-duplex formation of a single nucleic acid species in solution. We have adapted a technique utilizing a DNA nanoswitch that detects interaction of two different strands either with similar or differing lengths or modifications. Our method detected parallel duplex formation as a function of length, chemical modifications, and pH, and at a sensitivity that required over 100-fold less concentration of sample than prior UV-melting methods. While parallel polyriboadenylic acid and poly-2'-O-methyl-adenylic acid homo-duplexes formed, we did not detect homo-duplexes of polydeoxyriboadenylic acid strands or poly-locked nucleic acid (LNA)-adenylic strands. Importantly however, a poly-locked nucleic acid (LNA)-adenylic strand, as well as a poly-2'-O-methyl-adenylic strand, formed a hetero-duplex with a polyriboadenylic strand. Overall, our work validates a new tool for studying parallel duplexes and reveals fundamental properties of poly(A) parallel duplex formation. Parallel duplexes may find use in DNA nanotechnology and in molecular biology applications such as a potential poly(rA) tail capture tool as an alternative to traditional oligo(dT) based purification.


Assuntos
DNA/genética , Poli A/genética , Pareamento de Bases/genética , Conformação de Ácido Nucleico , Oligonucleotídeos/genética
15.
RNA ; 26(9): 1198-1215, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32424019

RESUMO

In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.


Assuntos
Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Mapas de Interação de Proteínas/genética , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Pareamento de Bases/genética , Catálise , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Mimetismo Molecular/genética , Estabilidade de RNA/genética
16.
Nucleic Acids Res ; 48(12): 6471-6480, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442296

RESUMO

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.


Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Simulação de Dinâmica Molecular , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Ligação de Hidrogênio , Mutação
17.
Nucleic Acids Res ; 48(11): 6234-6250, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396204

RESUMO

Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3' end of the syn-tasiRNA and the 5' end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Interferência de RNA , RNA Interferente Pequeno/genética , Pareamento de Bases , Sequência de Bases , Vetores Genéticos , RNA Interferente Pequeno/metabolismo , Tabaco/genética , Tabaco/virologia
18.
Nucleic Acids Res ; 48(11): 6294-6309, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402057

RESUMO

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.


Assuntos
Pareamento de Bases , Éxons , Íntrons , Processamento de RNA , Adenoviridae/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons/genética , Inativação Gênica , Íntrons/genética , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA/genética , Spliceossomos/metabolismo , Transcriptoma/genética , Globinas beta/genética
19.
Nucleic Acids Res ; 48(11): 5986-6000, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406922

RESUMO

Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11-13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.


Assuntos
Ilhas de CpG/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição , Alelos , Pareamento de Bases , Sequência de Bases , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Transcriptoma , Dedos de Zinco
20.
Nucleic Acids Res ; 48(11): 6120-6135, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421777

RESUMO

CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.


Assuntos
Adaptação Fisiológica/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , DNA/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Pareamento de Bases , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , DNA Helicases/metabolismo , Mutação , Hibridização de Ácido Nucleico , Plasmídeos/genética , Plasmídeos/metabolismo , Pyrococcus furiosus/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo
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