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Among RNA 5′-cap structures, γ-phosphate monomethylation is unique to a small subset of noncoding RNAs, 7SK and U6 in humans. 7SK is capped by methylphosphate capping enzyme (MePCE), which has a second nonenzymatic role as a core component of the 7SK ribonuclear protein (RNP), an essential regulator of RNA transcription. We report 2.0- and 2.1-Å X-ray crystal structures of the human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. 7SK recognition is achieved by protein contacts to a 5′-hairpin-single-stranded RNA region, thus explaining MePCE’s specificity for 7SK and U6. The structures reveal SAH and product RNA in a near-transition-state geometry. Unexpectedly, binding experiments showed that MePCE has higher affinity for capped versus uncapped 7SK, and kinetic data support a model of slow product release. This work reveals the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of 7SK RNP. Structural analysis of the human MePCE methyltransferase domain in complex with 7SK in the presence of SAH or SAM reveals that MePCE has higher affinity for capped 7SK and holds it for subsequent assembly of 7SK RNP.

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure–function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

The 5’ cap, catalyzed by RNA guanylyltransferase and 5’-phosphatase (RNGTT), is a vital mRNA modification for the functionality of mRNAs. mRNA capping occurs in the nucleus for the maturation of the functional mRNA and in the cytoplasm for fine-tuning gene expression. Given the fundamental importance of RNGTT in mRNA maturation and expression there is a need to further investigate the regulation of RNGTT. N6-methyladenosine (m 6 A) is one of the most abundant RNA modifications involved in the regulation of protein translation, mRNA stability, splicing, and export. We sought to investigate whether m 6 A could regulate the expression and activity of RNGTT. In this short report, we demonstrated that the 3’UTR of RNGTT mRNA is methylated with m 6 a by the m 6 A writer methyltransferase 3 (METTL3). Knockdown of METTL3 resulted in reduced protein expression of RNGTT. Sequencing of capped mRNAs identified an underrepresentation of ribosomal protein mRNA overlapping with 5’ terminal oligopyrimidine (TOP) mRNAs, and genes are dysregulated when cytoplasmic capping is inhibited. Pathway analysis identified disruptions in the mTOR and p70S6K pathways. A reduction in RPS6 mRNA capping, protein expression, and phosphorylation was detected with METTL3 knockdown

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These NSPs are found within open reading frame 1a (ORF1a) and open reading frame 1ab (ORF1ab) of the SARS-CoV-2 genome and encode NSP1 to NSP11 and NSP12 to NSP16, respectively. This study aimed to collect the available literature regarding NSP inhibitors. In addition, we searched the natural product database looking for similar structures. The results showed that similar structures could be tested as potential inhibitors of the NSPs.

Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2′,5′-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2′,5′-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L–mediated degradation. Thus, VP3-CTD is a 2′,5′-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2′,5′-PDEs that antagonize activation of innate immunity.

The 5′-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5′ termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5′-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5′ termini constitute a significant fraction of the human transcriptome. Hence, CMTr1–DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3'-to-5' exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3′-to-5′ exoribonuclease and the 2′-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication–transcription complex and virus replication.

At the end of my doctoral studies, in 1988, I published a review article on the major steps of transcription and translation during the mammalian reovirus multiplication cycle, a topic that still fascinates me 30 years later. It is in the nature of scientific research to generate further questioning as new knowledge emerges. Our understanding of these fascinating viruses thus remains incomplete but it seemed appropriate at this moment to look back and reflect on our progress and most important questions that still puzzle us. It is also essential of being careful about concepts that seem so well established, but could still be better validated using new approaches. I hope that the few reflections presented here will stimulate discussions and maybe attract new investigators into the field of reovirus research. Many other aspects of the viral multiplication cycle would merit our attention. However, I will essentially limit my discussion to these central aspects of the viral cycle that are transcription of viral genes and their phenotypic expression through the host cell translational machinery. The objective here is not to review every aspect but to put more emphasis on important progress and challenges in the field.