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2.
Curr Protoc ; 1(9): e248, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34529364

RESUMO

This article summarizes the protocols for phosphoramidite chemistry and solid phase synthesis of RNA oligonucleotides containing N4 -methylcytidine (m4 C) and N4 ,N4 -dimethylcytidine (m4 2 C) residues for base-pairing, structural, and enzymatic activity studies. The two key m4 C and m4 2 C phosphoramidite building blocks can be synthesized starting from the partially protected cytidine nucleosides, followed by solid-phase synthesis and HPLC purification of the modified target RNA oligonucleotides. These modified RNA strands are then prepared for base pairing stability, specificity, and structural studies using UV-melting temperature (Tm ) measurements and X-ray crystallography. Functional studies are performed by reverse transcription assays in primer extension reactions employing different enzymes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of m4 C phosphoramidite Basic Protocol 2: Synthesis of m4 2 C phosphoramidite Basic Protocol 3: Synthesis and purification of m4 C and m4 2 C containing RNA oligonucleotides.


Assuntos
Citidina , RNA , Pareamento de Bases , Nucleosídeos , Oligonucleotídeos
3.
Nat Commun ; 12(1): 5201, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465779

RESUMO

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.


Assuntos
Adenosina/análogos & derivados , DNA/química , DNA/metabolismo , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Pareamento de Bases , DNA/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Uridina/química , Uridina/genética , Uridina/metabolismo
4.
J Chem Phys ; 155(9): 095101, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34496591

RESUMO

Relative lifetimes of inherent double stranded DNA openings with lengths up to ten base pairs are presented for different gene promoters and corresponding mutants that either increase or decrease transcriptional activity in the framework of the Peyrard-Bishop-Dauxois model. Extensive microcanonical simulations are used with energies corresponding to physiological temperature. The bubble lifetime profiles along the DNA sequences demonstrate a significant reduction of the average lifetime at the mutation sites when the mutated promoter decreases transcription, while a corresponding enhancement of the bubble lifetime is observed in the case of mutations leading to increased transcription. The relative difference in bubble lifetimes between the mutated and wild type promoters at the position of mutation varies from 20% to more than 30% as the bubble length decreases.


Assuntos
DNA/genética , Pareamento de Bases , Sequência de Bases , DNA/química , Modelos Genéticos , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Transcrição Genética
5.
Biochemistry (Mosc) ; 86(8): 952-961, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488572

RESUMO

A-minor motifs are RNA tertiary structure motifs that generally involve a canonical base pair and an adenine base forming hydrogen bonds with the minor groove of the base pair. Such motifs are among the most common tertiary interactions in known RNA structures, comparable in number with the non-canonical base pairs. They are often found in functionally important regions of non-coding RNAs and, in particular, play a central role in protein synthesis. Here, we review local variations of the A-minor geometry and discuss difficulties associated with their annotation, as well as various structural contexts and common A-minor co-motifs, and diverse functions of A-minors in various processes in a living cell.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , RNA Bacteriano/metabolismo , RNA Catalítico/química , Ribossomos , Software
6.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34520542

RESUMO

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Assuntos
Algoritmos , RNA de Cadeia Dupla/química , RNA Mensageiro/química , RNA Viral/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Pareamento de Bases , Sequência de Bases , COVID-19/prevenção & controle , Humanos , Hidrólise , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Viral/genética , RNA Viral/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Termodinâmica
8.
Nat Commun ; 12(1): 4710, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354070

RESUMO

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.


Assuntos
DNA Bacteriano/genética , Genes Virais , Siphoviridae/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/metabolismo , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/genética , Adenilossuccinato Sintase/metabolismo , Bacteriófagos , Pareamento de Bases , Cristalografia por Raios X , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Viral , Redes e Vias Metabólicas , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Podoviridae/classificação , Podoviridae/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Siphoviridae/classificação , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445469

RESUMO

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.


Assuntos
Arabidopsis/enzimologia , Pareamento de Bases , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Arabidopsis/genética , Sítios de Ligação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Humanos , Especificidade por Substrato
10.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445396

RESUMO

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Deleção de Sequência , Pareamento de Bases , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , RNA/química , Ribonuclease III/genética
11.
Chemistry ; 27(56): 13991-13997, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34382264

RESUMO

The series of unnatural base pairs (UBPs) developed by the Romesberg lab, which pair via hydrophobic and packing interactions have been replicated, transcribed, and translated inside of a living organism. However, as to why these UBPs exhibit variable fidelity and efficiency when used in different contexts is not clear. In an effort to gain some insights, we investigated the thermal stability and pairing selectivity of the (d)NaM-(d)TPT3 UBP in 11nt duplexes via UV spectroscopy and the effects on helical structure via CD spectroscopy. We observed that while the duplexes containing a UBP are less stable than fully natural duplexes, they are generally more stable than duplexes containing natural mispairs. This work provides the first insights connecting the thermal stability of the (d)NaM-(d)TPT3 UBP to the molecular mechanisms for varying replication fidelity in different sequence contexts in DNA, asymmetrical transcription fidelity, and codon:anticodon interactions and can assist in future UBP development.


Assuntos
Pareamento de Bases
12.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445413

RESUMO

Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. In this study, using our Homology Segment Analysis software, we estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.


Assuntos
Cromatina/química , DNA Satélite/genética , Drosophila melanogaster/genética , Síndrome de Williams/genética , Cromossomo X/genética , Animais , Pareamento de Bases , Cromatina/genética , Biologia Computacional/métodos , Modelos Animais de Doenças , Humanos , Cromossomos Politênicos/genética , Software
13.
Nat Commun ; 12(1): 5114, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433819

RESUMO

Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.


Assuntos
Aprendizado Profundo , Biblioteca Gênica , Algoritmos , Pareamento de Bases , Edição de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
14.
J Chem Phys ; 154(19): 194102, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34240895

RESUMO

A statistical method is developed to estimate the maximum amplitude of the base pair fluctuations in a three dimensional mesoscopic model for nucleic acids. The base pair thermal vibrations around the helix diameter are viewed as a Brownian motion for a particle embedded in a stable helical structure. The probability to return to the initial position is computed, as a function of time, by integrating over the particle paths consistent with the physical properties of the model potential. The zero time condition for the first-passage probability defines the constraint to select the integral cutoff for various macroscopic helical conformations, obtained by tuning the twist, bending, and slide motion between adjacent base pairs along the molecule stack. Applying the method to a short homogeneous chain at room temperature, we obtain meaningful estimates for the maximum fluctuations in the twist conformation with ∼10.5 base pairs per helix turn, typical of double stranded DNA helices. Untwisting the double helix, the base pair fluctuations broaden and the integral cutoff increases. The cutoff is found to increase also in the presence of a sliding motion, which shortens the helix contour length, a situation peculiar of dsRNA molecules.


Assuntos
DNA/química , RNA/química , Pareamento de Bases , Modelos Moleculares
15.
J Phys Chem A ; 125(27): 5939-5955, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34228469

RESUMO

Repetitive nucleic acid sequences, which occur in abundance throughout the mammalian genome, are of enormous research interest due to their potential to adopt fascinating and unusual molecular structures such as the i-motif. In remarkable contrast to the DNA double helix, i-motif conformations are stabilized by protonated cytosine base pairs, (Cyt)H+(Cyt), that are centrally located in the core of the i-motif and intercalated vertically in an antiparallel fashion. An in-depth understanding of how modifications influence the stability of i-motif conformations is a prerequisite to understanding their biological functions and the development of effective means of tuning their stability for specific medical and technological applications. Here, the influence of the 2'- and 3'-hydroxy substituents of the sugar moieties and 5-methylation of the cytosine nucleobases on the base-pairing interactions of protonated cytidine nucleoside analogue base pairs, (xCyd)H+(xCyd), are examined by complementary threshold collision-induced dissociation techniques and computational methods. The xCyd nucleosides examined include the canonical DNA and RNA cytidine nucleosides, 2'-deoxycytidine (dCyd) and cytidine (Cyd), as well as several modified cytidine nucleoside analogues, 2',3'-dideoxycytidine (ddCyd), 5-methyl-2'-deoxycytidine (m5dCyd), and 5-methylcytidine (m5Cyd). Comparisons among these model base pairs indicate that the 2'- and 3'-hydroxy substituents of the sugar moieties have very little influence on the strength of the base-pairing interactions, whereas 5-methylation of the cytosine nucleobases is found to enhance the strength of the base-pairing interactions. The increase in stability resulting from 5-methylation is only modest but is more than twice as large for the DNA than RNA protonated cytidine base pair. Overall, present results suggest that canonical DNA i-motif conformations should be more stable than analogous RNA i-motif conformations and that 5-methylation of cytosine residues, a significant epigenetic marker, provides greater stabilization to DNA than RNA i-motif conformations.


Assuntos
Pareamento de Bases , Citidina/análogos & derivados , Metilação , Estrutura Molecular , Prótons , Termodinâmica
16.
J Chem Theory Comput ; 17(8): 4660-4674, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34270258

RESUMO

We introduce a method (FrD-LVC) based on a fragment diabatization (FrD) for the parametrization of a linear vibronic coupling (LVC) model suitable for studying the photophysics of multichromophore systems. In combination with effective quantum dynamics (QD) propagations with multilayer multiconfigurational time-dependent Hartree (ML-MCTDH), the FrD-LVC approach gives access to the study of the competition between intrachromophore decays, like those at conical intersections, and interchromophore processes, like exciton localization/delocalization and the involvement of charge-transfer (CT) states. We used FrD-LVC parametrized with time-dependent density functional theory (TD-DFT) calculations, adopting either CAM-B3LYP or ωB97X-D functionals, to study the ultrafast photoexcited QD of a guanine-cytosine (GC) hydrogen-bonded pair, within a Watson-Crick arrangement, considering up to 12 coupled diabatic electronic states and the effect of all of the 99 vibrational coordinates. The bright excited states localized on C and, especially, on G are predicted to be strongly coupled to the G → C CT state, which is efficiently and quickly populated after an excitation to any of the four lowest energy bright local excited states. Our QD simulations show that more than 80% of the excited population on G and ∼50% of that on C decay to this CT state in less than 50 fs. We investigate the role of vibronic effects in the population of the CT state and show that it depends mainly on its large reorganization energy so that it can occur even when it is significantly less stable than the bright states in the Franck-Condon region. At the same time, we document that the formation of the GC pair almost suppresses the involvement of dark nπ* excited states in the photoactivated dynamics.


Assuntos
Citosina/química , Guanina/química , Pareamento de Bases , Citosina/metabolismo , Teoria da Densidade Funcional , Guanina/metabolismo , Ligação de Hidrogênio
17.
Chem Commun (Camb) ; 57(60): 7438-7440, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34232244

RESUMO

Mechanical force applied by ultrasound in solution leads to the dissociation of DNA metallo-base-pair interactions when these motifs are functionalized with oligodeoxynucleotide sequences of sufficient length. The annealing and force-induced denaturing process is followed by the attachment of distance-sensitive fluorescent probes and is found to be reversible.


Assuntos
DNA/química , Oligodesoxirribonucleotídeos/química , Prata/química , Pareamento de Bases , DNA/genética , DNA/efeitos da radiação , Fluoresceínas/química , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico/efeitos da radiação , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/efeitos da radiação , Ondas Ultrassônicas
19.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34232998

RESUMO

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.


Assuntos
Aptâmeros de Nucleotídeos , COVID-19/virologia , Biblioteca Gênica , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , COVID-19/diagnóstico , Colorimetria/métodos , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros
20.
Nat Commun ; 12(1): 4545, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315864

RESUMO

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.


Assuntos
Íntrons/genética , Sítios de Splice de RNA/genética , Transcrição Genética , Pareamento de Bases/genética , Sequência de Bases , Éxons/genética , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcriptoma/genética
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