Q80289026
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The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.
- MeSH
- adenosin * analogy a deriváty metabolismus genetika analýza MeSH
- alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 * metabolismus genetika MeSH
- chromatografie kapalinová metody MeSH
- gen pro FTO * metabolismus genetika MeSH
- HEK293 buňky MeSH
- inosin * metabolismus genetika MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- messenger RNA * genetika metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.
- MeSH
- HIV infekce * virologie metabolismus MeSH
- HIV-1 * MeSH
- lidé MeSH
- malá jadérková RNA * metabolismus genetika MeSH
- NAD * metabolismus MeSH
- RNA čepičky metabolismus MeSH
- RNA malá jaderná * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- bakteriální RNA MeSH
- biochemie MeSH
- dinukleosidfosfáty MeSH
- Escherichia coli MeSH
- výzkum MeSH
- Publikační typ
- populární práce MeSH
- rozhovory MeSH
- MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- molekulární struktura MeSH
- organické sloučeniny křemíku chemická syntéza chemie farmakologie MeSH
- restrikční endonukleasy typu II antagonisté a inhibitory metabolismus MeSH
- štěpení DNA účinky léků MeSH
- stereoizomerie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of six pyrimidine-modified dNTPs--5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates--were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected restriction endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved.
A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.
- MeSH
- DNA sondy analýza chemie MeSH
- DNA vazebné proteiny chemie MeSH
- DNA-nukleotidylexotransferasa chemie MeSH
- elektrochemické techniky metody MeSH
- hybridizace nukleových kyselin metody MeSH
- molekulární struktura MeSH
- purinové nukleotidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification.
- MeSH
- denaturace nukleových kyselin MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA chemie metabolismus MeSH
- nukleosidy chemická syntéza chemie MeSH
- nukleotidy chemická syntéza chemie metabolismus MeSH
- Thermococcaceae enzymologie MeSH
- žlučové kyseliny a soli chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.
- MeSH
- barva MeSH
- barvení a značení metody MeSH
- DNA-dependentní DNA-polymerasy chemie MeSH
- DNA chemie MeSH
- elektrochemie MeSH
- luminiscence MeSH
- oligonukleotidy chemie MeSH
- osmium chemie MeSH
- oxidace-redukce MeSH
- reagencia zkříženě vázaná chemie MeSH
- ruthenium chemie MeSH
- Publikační typ
- práce podpořená grantem MeSH
