Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
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
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
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
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
