The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

• Klíčová slova
• Ap4A, Ap4A-RNA, Diadenosine Tetraphosphate, RNA Cap, RNA Modification,
• MeSH
• dinukleosidfosfáty * metabolismus   MeSH
• fosfatasy MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• NUDIX hydrolasy MeSH
• RNA čepičky * MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diadenosine tetraphosphate MeSH Prohlížeč
• dinukleosidfosfáty * MeSH
• fosfatasy MeSH
• NUDIX hydrolasy MeSH
• NUDT2 protein, human MeSH Prohlížeč
• RNA čepičky * MeSH

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• adenin chemie   metabolismus   MeSH
• aptamery nukleotidové chemická syntéza   genetika   MeSH
• cytosin chemie   metabolismus   MeSH
• deoxyribonukleosidy chemie   genetika   metabolismus   MeSH
• dinukleosidfosfáty chemie   genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• párování bází MeSH
• polymerázová řetězová reakce MeSH
• polymery chemická syntéza   metabolismus   MeSH
• replikace DNA * MeSH
• sekvence nukleotidů MeSH
• uracil chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• aptamery nukleotidové MeSH
• cytosin MeSH
• deoxyribonukleosidy MeSH
• dinukleosidfosfáty MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH
• polymery MeSH
• uracil MeSH

Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.

• MeSH
• bakteriofág T7 enzymologie   MeSH
• dinukleosidfosfáty genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• iniciace genetické transkripce * MeSH
• párování bází MeSH
• RNA čepičky genetika   MeSH
• RNA genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bacteriophage T7 RNA polymerase MeSH Prohlížeč
• dinukleosidfosfáty MeSH
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• RNA čepičky MeSH
• RNA MeSH
• virové proteiny MeSH

Dinucleotides (3'-5')-ApU and UpA and their 3'-O-phosphonylmethyl and 5'-O-phosphonylmethyl analogues were studied as substrates in the primed abortive synthesis catalysed by Escherichia coli DNA-dependent RNA polymerase on poly[d(A-T)] template. All phosphonate analogues of dinucleotides containing the anomalous sugar-phosphate backbone are substrates for the holoenzyme as verified by RNase A and RNase T2 digestion of the trinucleotide analogues obtained. The finding that phosphonate dinucleotides act as primers for transcription indicates that steric requirements at the initiation site are not as specific as previously supposed. Analysis of kinetic constants of ordered bibi reaction Kia, KmA, KmB and Vmax suggests that the instability of short RNA-DNA hybrids contributes to the abortive release of trinucleotides formed.

• MeSH
• dinukleosidfosfáty metabolismus   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• Escherichia coli enzymologie   MeSH
• genetická transkripce MeSH
• kinetika MeSH
• organofosfonáty MeSH
• poly dA-dT metabolismus   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dinukleosidfosfáty MeSH
• DNA řízené RNA-polymerasy MeSH
• organofosfonáty MeSH
• poly dA-dT MeSH