It was long time pressumed that only eukaryotes protect their RNA by 5'-RNA cap. Recently, it was shown that also prokaryotes employ some kind of protection of their RNA in the form of 5'-triphosphate or NAD covalently attached to 5'-terminus. This review discusses the state of art of 5'-RNA cap in eukaryotes and prokaryotes.

• Klíčová slova
• modifikace RNA,
• MeSH
• posttranskripční úpravy RNA MeSH
• RNA čepičky * MeSH
• výzkum MeSH
• Publikační typ
• práce podpořená grantem MeSH

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• endoribonukleasy metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• NAD metabolismus   MeSH
• nukleotidy genetika   MeSH
• počátek transkripce fyziologie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• RNA čepičky genetika   MeSH
• transkriptom genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

• MeSH
• bakteriální RNA genetika   MeSH
• dinukleosidfosfáty genetika   MeSH
• DNA řízené RNA-polymerasy genetika   MeSH
• Escherichia coli genetika   MeSH
• hydrolasy působící na anhydridy kyselin metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• metylace MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• RNA čepičky genetika   MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• polymerázová řetězová reakce MeSH
• ribonukleoproteiny MeSH
• RNA izolace a purifikace   MeSH
• sekvenční analýza RNA MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• molekulární biologie, molekulární medicína

Mpox is a zoonotic disease caused by the mpox virus (MPXV), which has gained attention due to its rapid and widespread transmission, with reports from more than 100 countries. The virus belongs to the Orthopoxvirus genus, which also includes variola virus and vaccinia virus. In poxviruses, the RNA cap is crucial for the translation and stability of viral mRNAs and also for immune evasion. This study presents the crystal structure of the mpox 2'-O-methyltransfarase VP39 in complex with a short cap-0 RNA. The RNA substrate binds to the protein without causing any significant changes to its overall fold and is held in place by a combination of electrostatic interactions, π-π stacking and hydrogen bonding. The structure also explains the mpox VP39 preference for a guanine base at the first position; it reveals that guanine forms a hydrogen bond that an adenine would not be able to form.

• MeSH
• lidé MeSH
• methyltransferasy chemie   MeSH
• metylace MeSH
• opičí neštovice * MeSH
• RNA čepičky * metabolismus   MeSH
• vazebná místa MeSH
• virové proteiny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.

• MeSH
• adenosin analogy a deriváty   metabolismus   farmakologie   MeSH
• Betacoronavirus enzymologie   MeSH
• chemické modely MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• katalytická doména MeSH
• koronavirové infekce virologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• methyltransferasy chemie   metabolismus   MeSH
• molekulární modely MeSH
• pandemie MeSH
• RNA čepičky MeSH
• RNA virová metabolismus   MeSH
• stabilita RNA MeSH
• virová pneumonie virologie   MeSH
• virové nestrukturální proteiny chemie   metabolismus   MeSH
• virové regulační a přídatné proteiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• cytoplazmatická granula metabolismus   MeSH
• intracelulární prostor MeSH
• konformace proteinů MeSH
• multiproteinové komplexy metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• ovarium růst a vývoj   metabolismus   MeSH
• proteiny vázající RNA fyziologie   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA čepičky metabolismus   MeSH
• RNA messenger skladovaná metabolismus   MeSH
• trans-aktivátory metabolismus   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• MeSH
• COVID-19 virologie   MeSH
• lidé MeSH
• methyltransferasy analýza   genetika   metabolismus   MeSH
• monoklonální protilátky analýza   MeSH
• RNA čepičky genetika   metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• SARS-CoV-2 chemie   enzymologie   genetika   MeSH
• transport proteinů MeSH
• virové nestrukturální proteiny analýza   genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny analýza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH