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.

• Keywords
• modifikace RNA,
• MeSH
• RNA Processing, Post-Transcriptional MeSH
• RNA Caps * MeSH
• Research MeSH
• Publication type
• Research Support, Non-U.S. Gov't 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-Directed RNA Polymerases metabolism   MeSH
• Endoribonucleases metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression genetics   MeSH
• Transcription, Genetic genetics   MeSH
• NAD metabolism   MeSH
• Nucleotides genetics   MeSH
• Transcription Initiation Site physiology   MeSH
• Promoter Regions, Genetic genetics   MeSH
• RNA Caps genetics   MeSH
• Transcriptome genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• RNA, Bacterial genetics   MeSH
• Dinucleoside Phosphates genetics   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Escherichia coli genetics   MeSH
• Acid Anhydride Hydrolases metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Methylation MeSH
• Escherichia coli Proteins metabolism   MeSH
• RNA Caps genetics   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Polymerase Chain Reaction MeSH
• Ribonucleoproteins MeSH
• RNA isolation & purification   MeSH
• Sequence Analysis, RNA MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• 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
• Humans MeSH
• Methyltransferases chemistry   MeSH
• Methylation MeSH
• Mpox, Monkeypox * MeSH
• RNA Caps * metabolism   MeSH
• Binding Sites MeSH
• Viral Proteins genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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 Infections * virology   metabolism   MeSH
• HIV-1 * MeSH
• Humans MeSH
• RNA, Small Nucleolar * metabolism   genetics   MeSH
• NAD * metabolism   MeSH
• RNA Caps metabolism   MeSH
• RNA, Small Nuclear * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article 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
• Adenosine analogs & derivatives   metabolism   pharmacology   MeSH
• Betacoronavirus enzymology   MeSH
• Models, Chemical MeSH
• Enzyme Inhibitors metabolism   pharmacology   MeSH
• Catalytic Domain MeSH
• Coronavirus Infections virology   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Methyltransferases chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Pandemics MeSH
• RNA Caps MeSH
• RNA, Viral metabolism   MeSH
• RNA Stability MeSH
• Pneumonia, Viral virology   MeSH
• Viral Nonstructural Proteins chemistry   metabolism   MeSH
• Viral Regulatory and Accessory Proteins chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Bacteriophage T7 enzymology   MeSH
• Dinucleoside Phosphates genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases genetics   metabolism   MeSH
• DNA metabolism   MeSH
• Transcription Initiation, Genetic * MeSH
• Base Pairing MeSH
• RNA Caps genetics   MeSH
• RNA genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Viral Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Cell Differentiation physiology   MeSH
• Cytoplasmic Granules metabolism   MeSH
• Intracellular Space MeSH
• Protein Conformation MeSH
• Multiprotein Complexes metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oocytes metabolism   MeSH
• Ovary growth & development   metabolism   MeSH
• RNA-Binding Proteins physiology   MeSH
• Ribonucleoproteins metabolism   MeSH
• RNA Caps metabolism   MeSH
• RNA, Messenger, Stored metabolism   MeSH
• Trans-Activators metabolism   MeSH
• Gene Expression Regulation, Developmental physiology   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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 virology   MeSH
• Humans MeSH
• Methyltransferases analysis   genetics   metabolism   MeSH
• Antibodies, Monoclonal analysis   MeSH
• RNA Caps genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• SARS-CoV-2 chemistry   enzymology   genetics   MeSH
• Protein Transport MeSH
• Viral Nonstructural Proteins analysis   genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins analysis   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH