RESUMO
MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.
Assuntos
MicroRNAs , Oligonucleotídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , Xenopus/genéticaRESUMO
Influenza A kills hundreds of thousands of people globally every year and has the potential to generate more severe pandemics. Influenza A's RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of 8 nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2 × 2-nt internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains 4 and likely 5 bp between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing.
Assuntos
Influenza Humana , Sítios de Splice de RNA , Sequência de Bases , Humanos , Influenza Humana/genética , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Oligonucleotídeos , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismoRESUMO
Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Humanos , Vírus da Influenza A Subtipo H1N1/genética , SARS-CoV-2/genética , Vírus da Influenza A/genética , RNA Viral/genética , GenômicaRESUMO
RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.
Assuntos
Reagentes de Ligações Cruzadas/química , Vírus da Influenza A/química , Influenza Humana/virologia , RNA Viral/química , Tiouridina/química , Pareamento de Bases , Sequência de Bases , Humanos , Conformação de Ácido NucleicoRESUMO
RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping the physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3'-terminus of the tetramolecular G-quadruplex. In this context, the U-tetrad adopts a unique conformation involving chain reversal and is responsible for a tremendous stabilization of the G-quadruplex (ΔTm up to 30°C). In this report, we attempt to rationalize the origin of this stabilizing effect by concurrent structural, thermal stability, and molecular dynamics studies of a series of G-quadruplexes with subtle chemical modifications at their 3'-termini. Our results provide detailed insights into the energetics of the "reversed" U-tetrad motif and the requirements for its formation. They point to the importance of the 2'OH to phosphate hydrogen bond and preferential stacking interactions for the formation propensity and stability of the motif.
Assuntos
Quadruplex G , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Uridina/química , Ligação de Hidrogênio , Simulação de Dinâmica MolecularRESUMO
Among types of trinucleotide repeats, there is some disproportion in the frequency of their occurrence in the human exome. This research presents new data describing the folding and thermodynamic stability of short, tandem RNA repeats of 23 types, focusing on the rare, yet poorly analyzed ones. UV-melting experiments included the presence of PEG or potassium and magnesium ions to determine their effect on the stability of RNA repeats structures. Rare repeats predominantly stayed single-stranded but had the potential for base pairing with other partially complementary repeat tracts. A coexistence of suitably complementary repeat types in a single RNA creates opportunities for interaction in the context of the secondary structure of RNA. We searched the human transcriptome for model RNAs in which different, particularly rare trinucleotide repeats coexist and selected the GABRA4 and CHIC1 RNAs to study intramolecular interactions between the repeat tracts that they contain. In vitro secondary structure probing results showed that the UAA and UUG repeat tracts, present in GABRA4 3' UTR, form a double helix, which separates one of its structural domains. For the RNA CHIC1 ORF fragment containing four short AGG repeat tracts and the CGU tract, we proved the formation of quadruplexes that blocked reverse transcription.
Assuntos
RNA , Repetições de Trinucleotídeos , Pareamento de Bases , Humanos , RNA/química , RNA/genética , TermodinâmicaRESUMO
Influenza A virus (IAV) is a member of the single-stranded RNA (ssRNA) family of viruses. The most recent global pandemic caused by the SARS-CoV-2 virus has shown the major threat that RNA viruses can pose to humanity. In comparison, influenza has an even higher pandemic potential as a result of its high rate of mutations within its relatively short (<13 kbp) genome, as well as its capability to undergo genetic reassortment. In light of this threat, and the fact that RNA structure is connected to a broad range of known biological functions, deeper investigation of viral RNA (vRNA) structures is of high interest. Here, for the first time, we propose a secondary structure for segment 8 vRNA (vRNA8) of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components. This structure shows similarities with prior in vitro experiments. Additionally, we determined the location of several well-defined, conserved structural motifs of vRNA8 within IAV strains with possible functionality. These RNA motifs appear to fold independently of regional nucleoprotein (NP)-binding affinity, but a low or uneven distribution of NP in each motif region is noted. This research also highlights several accessible sites for oligonucleotide tools and small molecules in vRNA8 in a cellular environment that might be a target for influenza A virus inhibition on the RNA level.
Assuntos
Regulação Viral da Expressão Gênica , Genoma Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Conformação de Ácido Nucleico , RNA Viral/química , Animais , Sequência de Bases , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Modelos Moleculares , Motivos de Nucleotídeos/genética , Dobramento de RNA , RNA Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Riboswitches alter gene expression in response to ligand binding, coupling sensing and regulatory functions to help bacteria respond to their environment. The structural determinants of ligand binding in the prequeuosine (7-aminomethyl-7-deazaguanine, preQ1) bacterial riboswitches have been studied, but the functional consequences of structural perturbations are less known. A new article combining biophysical and cell-based readouts of 15 mutants of the preQ1-II riboswitch from Lactobacillus rhamnosus demonstrates that ligand binding does not ensure successful gene regulation, providing new insights into these shapeshifting sequences.
Assuntos
Bactérias/genética , Lacticaseibacillus rhamnosus/genética , Riboswitch/genética , Bactérias/efeitos dos fármacos , Fenômenos Biofísicos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lacticaseibacillus rhamnosus/efeitos dos fármacos , Ligantes , Mutação/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Pirimidinonas/farmacologia , Pirróis/farmacologia , Riboswitch/efeitos dos fármacosRESUMO
Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high-resolution solution NMR structure of the r(UGGUGGU)4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad. Similar "reversed U-tetrads" were already observed in the crystalline phase. However, our NMR structure coupled with extensive explicit solvent molecular dynamics (MD) simulations identifies some key features of this motif that up to now remained overlooked. These include the presence of an exceptionally stable 2'OH to phosphate hydrogen bond, as well as the formation of an additional K+ binding pocket in the quadruplex groove.
Assuntos
Quadruplex G , Estabilidade de RNA , RNA/química , Sequência de Bases , Sítios de Ligação , Cátions/química , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Potássio/química , Espalhamento a Baixo Ângulo , Sódio/química , Uridina/química , Água/química , Difração de Raios XRESUMO
Influenza A virus (IAV) causes seasonal epidemics and sporadic pandemics, therefore is an important research subject for scientists around the world. Despite the high variability of its genome, the structure of viral RNA (vRNA) possesses features that remain constant between strains and are biologically important for virus replication. Therefore, conserved structural motifs of vRNA can represent a novel therapeutic target. Here, we focused on the presence of G-rich sequences within the influenza A/California/07/2009(H1N1) genome and their ability to form RNA G-quadruplex structures (G4s). We identified 12 potential quadruplex-forming sequences (PQS) and determined their conservation among the IAV strains using bioinformatics tools. Then we examined the propensity of PQS to fold into G4s by various biophysical methods. Our results revealed that six PQS oligomers could form RNA G-quadruplexes. However, three of them were confirmed to adopt G4 structures by all utilized methods. Moreover, we showed that these PQS motifs are present within segments encoding polymerase complex proteins indicating their possible role in the virus biology.
Assuntos
Quadruplex G , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Biologia Computacional , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The RNA "GAGU" duplex, (5'GACGAGUGUCA)2, contains the internal loop (5'-GAGU-3')2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans-Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'-endo conformation. This differs markedly from the internal loops, (5'-GAGC-3')2, (5'-AAGU-3')2, and (5'-UAGG-3')2, which all have cis-Watson-Crick/Watson-Crick AG "imino" pairs flanked by cis-Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-GAGC-3')2, (5'-AAGU-3')2, and (5'-UAGG-3')2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3')2, equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3')2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3')2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed.
Assuntos
Simulação de Dinâmica Molecular , RNA de Cadeia Dupla/química , Pareamento de Bases , Guanosina/química , Ligação de Hidrogênio , Inosina/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido NucleicoRESUMO
The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, α, ß, γ, and δ. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct δ, or general acid catalysis, via N3 of the syn adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the δ catalysis role of A1 via chemical rescue and the catalytic relevance of the syn orientation of the nucleobase by conformational analysis. Using inhibited twister ribozyme variants with A1(N3) deaza or A1 abasic modifications, we observe >100-fold chemical rescue effects in the presence of protonatable biological small molecules such as imidazole and histidine, similar to observed rescue values previously reported for C75U/C76Δ in the HDV ribozyme. Brønsted plots for the twister variants support a model in which small molecules rescue catalytic activity via a proton transfer mechanism, suggesting that A1 in the wild type is involved in proton transfer, most likely general acid catalysis. Additionally, through glycosidic conformational analysis in an appropriate background that accommodates the bromine atom, we observe that an 8BrA1-modified twister ribozyme is up to 10-fold faster than a nonmodified A1 ribozyme, supporting crystallographic data that show that A1 is syn when conducting proton transfer. Overall, this study provides functional evidence that the nucleotide immediately downstream of the cleavage site participates directly or indirectly in general acid-base catalysis in the twister ribozyme while occupying the syn conformation.
Assuntos
Glicosídeos/química , RNA Catalítico/química , Catálise , Concentração de Íons de Hidrogênio , Cinética , Modelos MolecularesRESUMO
Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the â¼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5' splice site (5'ss, located at the exon 10-intron 10 junction). These mutations tend to increase the 4R:3R ratio by destabilizing the hairpin, thereby improving 5'ss recognition by U1 snRNP. Interestingly, a single C-to-G mutation at the 19th nucleotide in intron 10 (C19G or +19G) decreases the level of exon 10 inclusion significantly from 56% to 1%, despite the disruption of a G-C base pair in the bottom stem of the hairpin. Here, we show by biophysical characterization, including thermal melting, fluorescence, and single-molecule mechanical unfolding using optical tweezers, that the +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability. The cell culture alternative splicing patterns of a series of minigenes reveal that the splicing activities of the mutants with destabilizing mutations on the top stem can be compensated in a position-dependent manner by the +19G mutation in the bottom stem. We observed an excellent correlation between the level of exon 10 inclusion and the rate of mechanical unfolding at 10 pN, indicating that the unfolding of the splice site hairpins (to facilitate subsequent binding of U1 snRNA) may be aided by helicases or other proteins.
Assuntos
Éxons , Splicing de RNA , RNA/química , Proteínas tau/genética , Sequência de Bases , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Mutação Puntual , RNA/genética , Dobramento de RNA , Temperatura de TransiçãoRESUMO
We report a case of Plasmodium knowlesi malaria imported to central Europe from Southeast Asia. Laboratory suspicion of P. knowlesi infection was based on the presence of atypical developmental forms of the parasite in Giemsa-stained microscopic smears. We confirmed and documented the clinical diagnosis by molecular biology techniques.
Assuntos
Malária/diagnóstico , Plasmodium knowlesi/isolamento & purificação , Adulto , Antimaláricos/uso terapêutico , Sudeste Asiático , Feminino , Humanos , Malária/tratamento farmacológico , Malária/microbiologia , Polônia , Reação em Cadeia da Polimerase , ViagemRESUMO
An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies.
Assuntos
Etilenoglicóis/química , Sequências Repetidas Invertidas , RNA Ligase (ATP)/química , RNA/química , Triazóis/química , Proteínas Virais/química , Bacteriófago T4/química , Pareamento de Bases , Sequência de Bases , Química Click , Ciclização , Éteres/química , Conformação de Ácido Nucleico , RNA/genética , RNA Ligase (ATP)/genética , Estabilidade de RNA , Termodinâmica , Proteínas Virais/genéticaRESUMO
Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10-intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10-15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3' arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.
Assuntos
Processamento Alternativo , Oligonucleotídeos Antissenso/genética , Ácidos Nucleicos Peptídicos/genética , Proteínas tau/genética , Éxons , Células HEK293 , Humanos , Conformação Molecular , Precursores de RNA , Sítios de Splice de RNA , RNA Mensageiro/genéticaRESUMO
Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a 'toxic' gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine ((Br)G)-modified molecule GC(Br)GGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GC(Br)GGCGGC)4 RNA quadruplex with (Br)G3:G6:(Br)G3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson-Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry.
Assuntos
Quadruplex G , RNA Mensageiro/química , Expansão das Repetições de Trinucleotídeos , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Expressão Gênica , Guanosina/análogos & derivados , Guanosina/química , Humanos , Modelos Moleculares , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Motivos de Nucleotídeos , RNA não Traduzido/química , AnimaisRESUMO
The influenza A virus (IAV) genome comprises eight negative-sense viral (v)RNA segments. The seventh segment of the genome encodes two essential viral proteins and is specifically packaged alongside the other seven vRNAs. To gain insights into the possible roles of RNA structure both within and without virions, a secondary structure model of a naked (protein-free) segment 7 vRNA (vRNA7) has been determined using chemical mapping and thermodynamic energy minimization. The proposed structure model was validated using microarray mapping, RNase H cleavage and comparative sequence analysis. Additionally, the detailed structures of three vRNA7 fragment constructs - comprising independently folded subdomains - were determined. Much of the proposed vRNA7 structure is preserved between IAV strains, suggesting their importance in the influenza replication cycle. Possible structure rearrangements, which allow or preclude long-range RNA interactions, are also proposed.
Assuntos
Vírus da Influenza A/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Genoma Viral/genética , Conformação de Ácido Nucleico , Ribonuclease H/metabolismo , TermodinâmicaRESUMO
Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure-function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.