Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 45.876
Filtrar
1.
Methods Mol Biol ; 2316: 287-312, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34845703

RESUMO

Viroids are considered the most minimalistic group of pathogens. Despite their presumed inability to encode for proteins, viroids induce several diseases in plants of primary economic importance. The production of viroid derived siRNAs (vd-siRNAs) of 21-24 nt, accompanies viroid infections in plants and results from the activation of the RNA silencing mechanism and specifically the function of Dicer endonucleases. A comprehensive set of experiments for the study and thorough analysis of viroid-infected plants has been developed. Here we present a detailed experimental plan including optimized protocols for plant infection by agroinfiltration, RNA extraction, and northern blot hybridization for the detection of both viroid genomic RNA and vd-siRNAs.


Assuntos
Viroides , Northern Blotting , Doenças das Plantas/genética , Plantas , Interferência de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , RNA Viral/genética , Viroides/genética
2.
Methods Mol Biol ; 2316: 97-109, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34845689

RESUMO

Northern blot analysis reveals information about RNA identity, size, and abundance. This technique has become an essential tool for the knowledge developed about viroids and also an excellent method for viroid detection. Here we describe the methodology of a Northern blot based in polyacrylamide gel electrophoresis under denaturing conditions, hybridized with a viroid full-length riboprobe labeled with chemiluminescence. Viroid detection with this approach entails positive signals, specific migration, and the differentiation of their circular and linear forms.


Assuntos
Viroides , Northern Blotting , Eletroforese em Gel de Poliacrilamida , Hibridização de Ácido Nucleico , RNA Viral/genética , Viroides/genética
3.
PLoS One ; 16(12): e0261476, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34932578

RESUMO

The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5' end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3' of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.


Assuntos
RNA Ribossômico 5,8S/análise , Northern Blotting , Linhagem Celular , Células HeLa , Humanos , RNA , Isoformas de RNA
4.
Methods Mol Biol ; 2298: 217-230, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085248

RESUMO

Queuosine (Q) is a hypermodified base that occurs at the wobble position of transfer RNAs (tRNAs) with a GUN anticodon. Q-tRNA modification is widespread among eukaryotes, yet bacteria are the original source of Q. Eukaryotes acquire Q from their diet, or from the gut microbiota (in multicellular organisms). Despite decades of study, the detailed roles of Q-tRNA modification remain to be elucidated, especially regarding its specific mechanisms of action. Here, we describe a method for the fast and reliable detection of Q-tRNA modification levels in individual tRNAs using a few micrograms of total RNA as starting material. The methodology is based on the co-polymerization of boronic acid (N-acryloyl-3-aminophenylboronic acid (APB)) in polyacrylamide gels, and on the interplay between this derivative and free cis-diol groups of the tRNA. During electrophoresis, the cis-diol groups slow down the Q-modified tRNA, which then can be separated from unmodified tRNA and quantified using Northern blot analysis.


Assuntos
Northern Blotting/métodos , Nucleosídeo Q/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Animais , Ácidos Borônicos/metabolismo , Humanos
5.
Methods Mol Biol ; 2348: 243-253, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160812

RESUMO

Viruses, like their metazoan hosts, have evolved to utilize intricate transcriptional mechanisms to generate a vast array of both coding and noncoding RNA transcripts. The resolution of specific noncoding RNA transcripts produced by viruses, particularly herpesviruses, presents a particularly difficult challenge due to their highly dense dsDNA genomes and their complex, overlapping, and context-dependent network of transcripts. While new long read sequencing platforms have facilitated the resolution of some noncoding transcripts from virus genomes, empirical molecular validation of transcripts from individual regions is essential. Herein, we demonstrate that the use of strand specific northern blots is essential for true validation of specific viral noncoding RNAs, and provide here a detailed molecular method for such an approach.


Assuntos
Northern Blotting , Homologia de Genes , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Viral/genética , Northern Blotting/métodos , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesviridae/genética , Fases de Leitura Aberta , Vírus/genética
6.
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
7.
Methods Mol Biol ; 2300: 41-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792870

RESUMO

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).


Assuntos
Reagentes para Ligações Cruzadas/química , Sondas de DNA/metabolismo , Etildimetilaminopropil Carbodi-Imida/química , RNA/análise , Northern Blotting , Sondas de DNA/química , Eletroforese em Gel de Gradiente Desnaturante , Digoxigenina/química , Eletroforese em Gel de Poliacrilamida Nativa , RNA/química
8.
Methods Mol Biol ; 2300: 65-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792872

RESUMO

Discovery and characterization of microRNAs (miRNAs) and other families of small RNAs lead researchers to study their structures/functions and their expression patterns. The splinted ligation method described here is based on nucleic acid hybridization. It is optimized for the direct labeling and quantitative detection of small RNAs. A specific bridge DNA oligonucleotide is used, which is perfectly complementary to both the target small RNA and a labeled ligation nucleic acid. The target RNA is subsequently labeled by ligation, detected by analysis in denaturing conditions, and quantified by phosphorimaging. The protocol does not require any specific material, and the procedure is fast and sensitive.


Assuntos
MicroRNAs/análise , MicroRNAs/química , Sondas de Oligonucleotídeos/metabolismo , Northern Blotting , DNA Ligases/metabolismo , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/química , Coloração e Rotulagem
9.
Nat Commun ; 12(1): 2316, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875662

RESUMO

Synthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.


Assuntos
Autofagia/genética , Estabilidade de RNA/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Vacúolos/genética , Northern Blotting , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
10.
Methods Mol Biol ; 2244: 301-342, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555594

RESUMO

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to detect, express and inhibit miRNAs, as well as methods used to identify and validate their targets, specifically with respect to the miRNAs encoded by human cytomegalovirus.


Assuntos
Citomegalovirus/genética , Imunoprecipitação/métodos , MicroRNAs/análise , Regiões 3' não Traduzidas/genética , Northern Blotting/métodos , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Humanos , MicroRNAs/genética , RNA Mensageiro/genética
11.
Cold Spring Harb Protoc ; 2021(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397778

RESUMO

RNA samples that have been transferred and fixed to a membrane may be hybridized with a specific probe to locate the RNA species of interest. Any one of a large number of methods can be used to label and detect probes, at the discretion of the investigator. After treating the membrane with blocking agents that suppress nonspecific absorption of the probe, the membrane is incubated under conditions that favor hybridization of the labeled probe to the immobilized target RNA. The membrane is then washed extensively to remove adventitiously bound probe and finally manipulated to yield an image of the distribution of the tightly bound probe on the membrane. After analysis of the results, the probe may be stripped from the membrane, and the membrane can then be used again in another hybridization experiment.


Assuntos
Northern Blotting/métodos , Hibridização de Ácido Nucleico/métodos , Radioatividade
12.
Methods Mol Biol ; 2174: 89-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813246

RESUMO

With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75% of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. Here, we provide a collection of detailed experimental protocols for lncRNA studies, including lncRNA immunoprecipitation, lncRNA pull-down, lncRNA northern blot analysis, lncRNA in situ hybridization, and lncRNA knockdown. We hope that the information included in this chapter can speed up research on lncRNAs biology and eventually lead to the development of clinical applications with lncRNA as novel prognostic markers and therapeutic targets.


Assuntos
Biologia Molecular/métodos , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Biomarcadores Tumorais/genética , Northern Blotting/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Genoma Humano , Humanos , Imunoprecipitação/métodos , Hibridização In Situ/métodos , Neoplasias/patologia , Oncogenes , RNA Longo não Codificante/metabolismo , Transdução de Sinais
13.
Methods Mol Biol ; 2167: 27-44, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712913

RESUMO

Retrozymes are a novel family of non-autonomous retrotransposable elements that contain hammerhead ribozyme motifs. These retroelements are found widespread in eukaryotic genomes, with active copies present in many species, which rely on other autonomous transposons for mobilization. Contrary to other retrotransposons, transcription of retrozymes in vivo leads to the formation and accumulation of circular RNAs, which can be readily detected by RNA blotting. In this chapter, we describe the procedures needed to carry out the cloning of genomic retrozymes, and to detect by northern blot their circular RNA retrotransposition intermediates.


Assuntos
Northern Blotting/métodos , Clonagem Molecular/métodos , RNA Catalítico/genética , RNA Catalítico/isolamento & purificação , RNA Circular/genética , Retroelementos/genética , Animais , Genoma , Motivos de Nucleotídeos , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , RNA Catalítico/metabolismo , RNA Circular/metabolismo
14.
Methods Mol Biol ; 2167: 205-224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712922

RESUMO

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleases/metabolismo , Edição de Genes/métodos , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , RNA Catalítico/metabolismo , RNA Guia/genética , Proteínas de Bactérias/genética , Northern Blotting , Endonucleases/genética , Endonucleases/metabolismo , Ensaios Enzimáticos/métodos , Citometria de Fluxo , Técnicas de Inativação de Genes , Inativação Gênica , Genes Reporter , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Mutação INDEL , Luciferases , RNA Catalítico/genética , RNA Guia/metabolismo , Receptores CCR5/genética
15.
Methods Mol Biol ; 2167: 225-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712923

RESUMO

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.


Assuntos
Proteínas Argonauta/genética , Inativação Gênica , Infecções por HIV/genética , HIV-1/genética , Vírus Delta da Hepatite/genética , RNA Catalítico/genética , RNA Guia/genética , RNA Interferente Pequeno/genética , Proteínas Argonauta/metabolismo , Northern Blotting , Clonagem Molecular/métodos , Ensaios Enzimáticos/métodos , Vetores Genéticos , Células HEK293 , HIV-1/metabolismo , Humanos , Sequências Repetidas Invertidas/genética , Luciferases/metabolismo , Interferência de RNA , RNA Catalítico/metabolismo , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/virologia , Transfecção/métodos
16.
Methods Mol Biol ; 2167: 253-267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712924

RESUMO

RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.


Assuntos
Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/isolamento & purificação , Clonagem Molecular/métodos , Reação em Cadeia da Polimerase/métodos , RNA Catalítico/genética , RNA Catalítico/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Northern Blotting , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Células HEK293 , Humanos , RNA Catalítico/química , Pequeno RNA não Traduzido/isolamento & purificação
17.
Methods Mol Biol ; 2167: 271-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712925

RESUMO

A lariat cap is a naturally occurring substitute of a conventional mRNA cap and is found in a particular genomic setting in a few eukaryotic microorganisms. It is installed by the lariat capping ribozyme acting in cis. In principle, any RNA molecule in any organism can be equipped with a lariat cap in vivo when expressed downstream of a lariat capping ribozyme. Lariat capping is thus a versatile tool for studying the importance of the 5' end structure of RNA molecules. In this chapter, we present protocols to validate the presence of the lariat cap and measure the efficiency of in vivo cleavage by the lariat capping ribozyme.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Leveduras/metabolismo , Northern Blotting , Fosfatos de Dinucleosídeos/genética , Eletroforese em Gel de Poliacrilamida , Exonucleases/metabolismo , Citometria de Fluxo , Sítios Internos de Entrada Ribossomal/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/metabolismo , RNA Mensageiro/química , Leveduras/genética
18.
Methods Mol Biol ; 2170: 45-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797450

RESUMO

Northern blotting is a classical technique that allows the detection of specific nucleic acids using radioactive or non-radioactive probes. Normally, nucleic acids are denatured and separated by agarose or polyacrylamide gel electrophoresis and transferred and fixed to a membrane prior to detection. Here, we describe a method to analyze specific RNA in native ribonucleoprotein complexes using blue native PAGE with subsequent northern blotting, crosslinking of RNA onto a suitable membrane, and detection using non-radioactive probes.


Assuntos
Northern Blotting/métodos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo
19.
Methods Mol Biol ; 2170: 155-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797458

RESUMO

Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years it has been aptly adapted to validate and study the size and expression of small noncoding RNAs. Here, we describe protocols employed in our laboratory for conventional northern analysis with total RNA/mRNA to study gene expression and validation of small noncoding RNAs using low molecular weight fraction of RNAs. A brief account on the recent advancements for improving the sensitivity and efficiency of northern blot detection is also included in this chapter.


Assuntos
Northern Blotting/métodos , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética
20.
J Alzheimers Dis ; 79(2): 793-806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33337366

RESUMO

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia caused by irreversible neurodegeneration, with the onset mechanisms elusive. tRNA-derived RNA fragments (tRFs), a recently discovered family of small non-coding RNAs (sncRNAs), have been found to associate with many human diseases, including infectious, metabolic, and neurological diseases. However, whether tRFs play a role in human AD development is not known. OBJECTIVE: This study aimed to explore whether tRFs are involved in human AD. METHODS: Thirty-four postmortem human hippocampus samples were used. The expression of Drosha, Dicer, and angiogenin (ANG), three ribonucleases responsible for the biogenesis of sncRNAs, was determined by qRT-PCR and western blot. The tRFs in the hippocampus was detected by qRT-PCR or northern blot. We also used qRT-PCR to quantify NOP2/Sun RNA methyltransferase 2 (NSun2) and polyadenylation factor I subunit 1 (CLP1), two tRNA modification enzymes. RESULTS: tRFs derived from a subset of tRNAs are significantly altered in the hippocampus of AD patients. The expression change of some tRFs showed age- and disease stage-dependent. ANG is significantly enhanced in AD, suggesting its role in inducing tRFs in AD. The expression of NSun2 in AD patients younger than 65 was significantly decreased. According to a previous report supporting NSun2-mediated tRNA methylation modification making tRNA less susceptible to ANG-mediated cleavage, our results suggested that the decrease in NSun2 may make tRNAs less methylated and subsequently enhanced tRF production from ANG-mediated tRNA cleavage. CONCLUSION: Our studies demonstrated for the first time the involvement of tRFs in human AD.


Assuntos
Doença de Alzheimer/diagnóstico , Hipocampo/metabolismo , RNA de Transferência/metabolismo , Idoso , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Biomarcadores , Northern Blotting , Estudos de Casos e Controles , Humanos , Reação em Cadeia da Polimerase , Pequeno RNA não Traduzido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...