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1.
Anal Chem ; 94(9): 4119-4125, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35195982

RESUMO

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.


Assuntos
Sistemas CRISPR-Cas , Reação em Cadeia da Ligase , MicroRNAs , Sondas RNA , Sistemas CRISPR-Cas/genética , Reação em Cadeia da Ligase/métodos , MicroRNAs/análise , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sondas RNA/genética , RNA Guia/genética
2.
Anal Chem ; 94(2): 960-967, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34989563

RESUMO

Mitochondrial membrane potential (ΔΨm) is an important biophysical parameter playing central roles in cell apoptosis, mitochondrial dysfunction, and other biological and pathological processes. Herein, we have rationally designed and fabricated a unique fluorescent probe for convenient ΔΨm visualization based on hot-band absorption and controllable anti-Stokes shift emission. The robust probe was excitable via hot-band absorption and emitted anti-Stokes upconversion emission and Stokes downconversion fluorescence simultaneously. The anti-Stokes emission could be efficiently inhibited upon the binding to RNA. The cationic probe targeted mitochondria in living cells with high ΔΨm and displayed both anti-Stokes green emission and ordinary red fluorescence. After the decrease of ΔΨm, the probe immigrated out of mitochondria to RNA and nucleolus, which showed only red emission owing to the inhibition of anti-Stokes fluorescence. In this manner, the ΔΨm could be visualized in dual-color mode. The probe enabled clearly monitoring the reversible changes in ΔΨm and was successfully employed to visualize oxidative damage of living cells. The decrease of ΔΨm in living tissues was also successfully observed with the newly designed probe.


Assuntos
Corantes Fluorescentes , Mitocôndrias , Apoptose , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Sondas RNA
3.
ACS Sens ; 7(1): 50-59, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-34985283

RESUMO

Detecting RNA at single-nucleotide resolution is a formidable task. Plasmodium falciparum is the deadliest form of malaria in humans and has shown to gain resistance to essentially all antimalarial drugs including artemisinin and chloroquine. Some of these drug resistances are associated with single-nucleotide polymorphisms (SNPs). Forced-intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that are designed as RNA-sensing molecules that fluoresce upon hybridization to their complementary (RNA) targets. We have previously designed and synthesized FIT-PNAs that target the C580Y SNP in the K13 gene of P. falciparum. In addition, we have now prepared FIT-PNAs that target the K76T SNP in the CRT gene of P. falciparum. Both SNPs are common ones associated with artemisinin and chloroquine drug resistance, respectively. Our FIT-PNAs are conjugated to a simple cell-penetrating peptide (CPP) that consists of eight d-lysines (dK8), which renders these FIT-PNAs cell-permeable to infected red blood cells (iRBCs). Herein, we demonstrate that FIT-PNAs clearly discriminate between wild-type (WT) strains (NF54-WT: artemisinin-sensitive or chloroquine-sensitive) and mutant strains (NF54-C580Y: artemisinin-resistant or Dd2: chloroquine-resistant) of P. falciparum parasites. Simple incubation of FIT-PNAs with live blood-stage parasites results in a substantial difference in fluorescence as corroborated by FACS analysis and confocal microscopy. We foresee FIT-PNAs as molecular probes that will provide a fast, simple, and cheap means for the assessment of drug resistance in malaria─a tool that would be highly desirable for the optimal choice of antimalarial treatment in endemic countries.


Assuntos
Antimaláricos , Malária Falciparum , Ácidos Nucleicos Peptídicos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Humanos , Ácidos Nucleicos Peptídicos/farmacologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/uso terapêutico , RNA , Sondas RNA
4.
Nucleic Acids Res ; 50(1): e3, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34591964

RESUMO

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3'-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5' ends (including m7G cap) in high yields (70-100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3'-end labelling with 5'-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.


Assuntos
Corantes Fluorescentes/metabolismo , Sondas RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Células HeLa , Humanos , Peixe-Zebra
5.
Mol Ecol Resour ; 22(3): 891-907, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34582623

RESUMO

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing extremely low endogenous content (<1%). Performance was, however, more limited for those samples already characterized by good DNA preservation (>20%-30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensitive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypomethylated regions, which improved data quality by reducing post-mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.


Assuntos
DNA Antigo , RNA , Animais , Automação , Cavalos/genética , RNA/genética , Sondas RNA , Análise de Sequência de DNA/métodos
6.
J Appl Microbiol ; 132(2): 1526-1542, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34424588

RESUMO

AIMS: Aerobic methane oxidation coupled to denitrification (AME-D) is a promising process for removing nitrate from groundwater and yet its microbial mechanism and ecological implications are not fully understood. This study used RNA stable isotope probing (RNA-SIP) and high-throughput sequencing to identify the micro-organisms that are actively involved in aerobic methane oxidation within a denitrifying biofilm. METHODS AND RESULTS: Two RNA-SIP experiments were conducted to investigate labelling of RNA and methane monooxygenase (pmoA) transcripts when exposed to 13 C-labelled methane over a 96-hour time period and to determine active bacteria involved in methane oxidation in a denitrifying biofilm. A third experiment was performed to ascertain the extent of 13 C labelling of RNA using isotope ratio mass spectrometry (IRMS). All experiments used biofilm from an established packed bed reactor. IRMS confirmed 13 C enrichment of the RNA. The RNA-SIP experiments confirmed selective enrichment by the shift of pmoA transcripts into heavier fractions over time. Finally, high-throughput sequencing identified the active micro-organisms enriched with 13 C. CONCLUSIONS: Methanotrophs (Methylovulum spp. and Methylocystis spp.), methylotrophs (Methylotenera spp.) and denitrifiers (Hyphomicrobium spp.) were actively involved in AME-D. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use RNA-SIP and high-throughput sequencing to determine the bacteria active within an AME-D community.


Assuntos
Metano , Microbiota , Biofilmes , Sequenciamento de Nucleotídeos em Larga Escala , Isótopos , Microbiota/genética , Oxirredução , Filogenia , RNA , Sondas RNA , RNA Ribossômico 16S
7.
Methods Mol Biol ; 2450: 359-371, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359318

RESUMO

Scleractinians, or stony corals, are colonial animals that possess a high regenerative capacity and a highly diverse innate immune system. As such they present the opportunity to investigate the interconnection between regeneration and immunity in a colonial animal. Understanding the relationship between regeneration and immunity in stony corals is of further interest as it has major implications for coral reef health. One method for understanding the role of innate immunity in scleractinian regeneration is in situ hybridization using RNA probes. Here we describe a protocol for in situ hybridization in adult stony corals using a digoxigenin (DIG)-labeled RNA antisense probe which can be utilized to investigate the spatial expression of immune factors during regeneration.


Assuntos
Antozoários , Animais , Antozoários/genética , Antozoários/metabolismo , Digoxigenina/metabolismo , Expressão Gênica , Hibridização In Situ , Sondas RNA/metabolismo
8.
Cold Spring Harb Protoc ; 2022(3)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34907077

RESUMO

In molecular cloning, digoxigenin is used as a ligand that can be incorporated into DNA and RNA probes and detected after hybridization with an anti-digoxigenin-antibody enzyme conjugate. Methods to label nucleic acids with digoxigenin and to detect digoxigenin-labeled probes are introduced here.


Assuntos
DNA , Ácidos Nucleicos , Digoxigenina , Hibridização de Ácido Nucleico , Sondas RNA
9.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34854870

RESUMO

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.


Assuntos
Imagem Molecular , RNA Polimerase II/metabolismo , Sondas RNA/metabolismo , Transcrição Genética , Núcleo Celular/metabolismo , Sobrevivência Celular , Células HeLa , Humanos , Interfase , Fosforilação , Fosfosserina/metabolismo
10.
Methods Mol Biol ; 2316: 89-96, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34845688

RESUMO

A simplified dot-blot hybridization protocol for Potato spindle tuber viroid (PSTVd) detection in Solanaceae species is described here. The protocol uses an RNA DIG-labeled probe and a simplified extraction procedure that avoids the use of hazardous chemicals. PSTVd was detected in composite tomato leaf samples in a ratio of up to 1:15 of PSTVd-infected to non-infected tissue and in composite potato tuber samples in a ratio up to 1:5 of PSTVd-infected to non-infected tissue. In Brugmansia spp., PSTVd was detected solely in the standard sample extract preparation. The method is suitable for a reliable, large-scale sample screening especially where cost is a limiting factor.


Assuntos
Solanum tuberosum , Viroides , Lycopersicon esculentum , Hibridização de Ácido Nucleico , Doenças das Plantas , Sondas RNA , RNA Viral/genética , Viroides/genética
11.
Methods Mol Biol ; 2403: 33-42, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34913114

RESUMO

Analysis of animal models allows a deeper understanding of craniofacial development in health and diseases of humans. Wholemount in situ hybridization (WISH) is an informative technique to visualize gene expression in tissues across the developmental stages of embryos. The principle of WISH is based on the complementary binding (hybridization) of the DNA/RNA probe to the target transcript. The bound probe can then be visualized by an enzymatic color reaction to delineate the expression pattern of transcripts within a tissue. Here we describe an optimized method to perform in situ hybridization in mouse embryos.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Animais , Expressão Gênica , Hibridização In Situ , Camundongos , Sondas RNA
12.
Methods Mol Biol ; 2404: 299-310, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694616

RESUMO

RNA has an extraordinary capacity to fold and form intrinsic secondary structures that play a central role in maintaining its functionality. It is crucial to have ways to study RNA structures and identify their functions in their biological environment. In the last few decades, a number of different chemical probing methods have been used to study RNA secondary structure. Here, we present a dimethyl sulfate-based (DMS) chemical probing method coupled with Next Generation sequencing (DMS-MaPseq) to study RNA secondary structure in vivo.DMS modifies unpaired adenine and cytosine bases which are then converted to mutations/mismatches using a thermostable group II intron reverse transcriptase (TGIRT) and further analyzed using sequencing. We validated the technique in model systems ranging from Drosophila to human cell lines, thus increasing the technique's broad range of applications. DMS-MaPseq provides high quality data and can be used for both gene-targeted as well as genome-wide analysis.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Animais , Humanos , RNA/genética , Sondas RNA , Análise de Sequência de RNA , Ésteres do Ácido Sulfúrico
13.
Adv Mater ; 33(45): e2103131, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34541724

RESUMO

Organelles are specialized compartments, where various proteins reside and play crucial roles to maintain essential cellular structures and functions in mammalian cells. A comprehensive understanding of protein expressions and subsequent localizations at each organelle is of great benefit to the development of organelle-based therapies. Herein, a set of single or dual organelle labeling messenger RNAs (SOLAR or DOLAR) is designed as novel imaging probes, which encode fluorescent proteins with various organelle localization signals. These mRNA probes enable to visualize the protein localizations at different organelles and investigate their trafficking from ribosomal machinery to specific organelles. According to the in vitro results, SOLAR probes show organelle targeting capabilities consistent with the design. Moreover, DOLAR probes with different linkers display distinct targeting properties depending on different organelle localization signals. Additionally, these mRNA probes also exhibit organelle labeling ability in vivo when delivered by lipid nanoparticles (LNPs). Therefore, these mRNA-based probes provide a unique tool to study cell organelles and may facilitate the design of organelle-based therapies.


Assuntos
Lipossomos/química , Nanopartículas/química , Organelas/química , Sondas RNA/química , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Lisossomos/metabolismo , Camundongos , Microscopia Confocal , Organelas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sondas RNA/metabolismo , RNA Mensageiro/química
14.
Anal Bioanal Chem ; 413(28): 6963-6971, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34581826

RESUMO

An efficient electrogenerated chemiluminescence (ECL) nanoprobe (luminol-Au NPs-Ti3C2) was constructed based on Ti3C2Tx MXene (Ti3C2)-mediated in situ formation of Au NPs and anchoring luminol to fabricate a sensitive ECL biosensor for miRNA-155 detection. Herein, Ti3C2 with rich Ti vacancy defects was used as reducing agent, and Au NPs were generated in situ and anchored on the Ti3C2 (Au NPs-Ti3C2). Moreover, the Au NPs-Ti3C2 composites were used as a carrier and provided a large number of sites for the efficient linking of luminol through Au-N bonds to form stable luminol-Au NPs-Ti3C2. The immobilization of ECL emitters is a versatile strategy which not only shortens the electron transmission distance between luminol and electrode, but also provides naked catalytic predominated (111) facets of Au NPs with high electrocatalytic activity, significantly improving the ECL signal of luminol. Furthermore, a catalytic hairpin assembly (CHA) reaction was used, resulting in further amplification of the signal. As a result, the as-prepared ECL biosensor exhibited a linear range from 0.3 fM to 1 nM with a detection limit of 0.15 fM, and demonstrated high reliability of miRNA-155 detection even in human serum samples. The construction of a multifunctional ECL probe with excellent ECL emission opens a new chapter for the application of Ti3C2 in the field of bioanalysis. Herein, Au NPs were generated in situ and anchored on the Ti3C2 (Au NPs-Ti3C2). Moreover, the Au NPs-Ti3C2 was used as a carrier and linked luminol through Au-N bonds to form a stable luminol-Au NPs-Ti3C2 nanoprobe. The strategy displayed versatility which not only shortened the electron transmission distance between luminol and the electrode, but also provided a catalytic surface with high electrocatalytic activity of Au NPs that significantly improved the ECL signal of luminol.


Assuntos
Ouro/química , Luminescência , Luminol/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Sondas RNA/química , Titânio/química , Técnicas Biossensoriais/métodos , Eletroforese em Gel de Poliacrilamida , Humanos , Reprodutibilidade dos Testes , Análise Espectral/métodos
15.
Viruses ; 13(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209211

RESUMO

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.


Assuntos
Norovirus/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Alanina/genética , Aminoácidos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Norovirus/genética , Ligação Proteica , Sondas RNA
16.
STAR Protoc ; 2(3): 100647, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34278333

RESUMO

Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in Saccharomyces cerevisiae with optimized lyticase digestion time and hybrization steps for more homogenous results. For complete details on the use and execution of this protocol, please refer to Donovan et al. (2019).


Assuntos
Hibridização in Situ Fluorescente/métodos , Imagem Molecular/métodos , Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos , Regulação Fúngica da Expressão Gênica , Sondas RNA/genética , RNA Fúngico
17.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
18.
Methods Mol Biol ; 2324: 187-202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165716

RESUMO

One of the most commonly described biological feature of processed pseudogenes is the ability to influence the expression of their parental coding genes. As evidenced in several studies, the high sequence similarity between these RNA pairs sets up a certain level of competition for posttranscriptional regulators, including, among others, RNA-binding proteins (RBPs). RBPs may affect, positively or negatively, the stability of bound mRNAs, so that, if an overexpressed pseudogene competes with its homologous coding gene, the downstream protein synthesis would change, with potential pathological consequences. Given these premises, a rigorous and comprehensive understanding of interactions between pseudogene-parental gene RNA pairs and RBPs could provide further insights into the biological bases of complex diseases, such as cancer, cardiovascular disease, and type 2 diabetes, identifying novel predictive and/or prognostic biomarkers.Herein, we detail easily adaptable protocols of plasmid-based molecular cloning and RNA-electrophoretic mobility shift assay (EMSA) used in our laboratory for determining the interaction between a cytoplasmatic stabilizing protein (αCP1) and the pseudogene-parental gene RNA pair HMGA1-p /HMGA1. We also offer a general overview of RNA immunoprecipitation procedures and present novel bioinformatic tools for predicting RBPs binding sites on pseudogene transcripts.


Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Imunoprecipitação/métodos , Pseudogenes/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sítios de Ligação , Ligação Competitiva , Biotinilação , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Proteína HMGA1a/genética , Humanos , Medições Luminescentes , Ligação Proteica , Sondas RNA , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transfecção
19.
Methods Mol Biol ; 2348: 371-383, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160818

RESUMO

Circular RNAs (circRNAs) are covalently closed transcripts generated by back-splicing reaction. The lack of free ends endows these RNA molecules with high stability thus allowing them to accumulate in tissues and body fluids. They are widely expressed in most organisms, are modulated during development and display tissue-specific expression, resulting particularly enriched in the nervous system. Deregulation of circRNA expression has also been associated with several pathological conditions including neurological diseases and cancer.Here we present a Northern blot procedure that allows the analysis of the expression of bona fide circRNAs through the use of a digoxigenin-labeled RNA probe and the immunodetection of the signals.


Assuntos
Northern Blotting/métodos , Expressão Gênica , RNA Circular , Humanos , Sondas RNA , RNA não Traduzido
20.
Cold Spring Harb Protoc ; 2021(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941667

RESUMO

In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.


Assuntos
Ensaios de Proteção de Nucleases/métodos , Sondas RNA , RNA/análise , Ribonucleases , Hibridização de Ácido Nucleico , RNA/química , RNA/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/química , Ribonuclease T1/metabolismo , Ribonuclease Pancreático/metabolismo
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