The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Generation and Characterization of In Vitro Transcribed mRNA.

Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Towards T7 RNA polymerase (T7RNAP)-based expression system in yeast: challenges and opportunities.

During the last decades, we have witnessed unprecedented advances in biological engineering and synthetic biology. These disciplines aim to take advantage of gene pathway regulation and gene expression in different organisms, to enable cells to perform desired functions. Yeast has been widely utilized as a model for the study of eukaryotic protein expression while bacteriophage T7RNAP and its promoter constitute the preferred system for prokaryotic protein expression (such as pET-based expression systems). The ability to integrate a T7RNAP-based expression system in yeast could allow for a better understanding of gene regulation in eukaryotic cells, and potentially increase the efficiency and processivity of yeast as an expression system. However, the attempts for the creation of such a system have been unsuccessful to date. This review aims to: (i) summarize the efforts that, for many years, have been devoted to the creation of a T7RNAP-based yeast expression system and ii) provide an overview of the latest advances in knowledge of eukaryotic transcription and translation that could lead to the construction of a successful T7RNAP expression system in yeast. The completion of this new expression system would allow to further expand the toolkit of yeast in synthetic biology and ultimately contribute to boost yeast usage as a key cell factory in sustainable biorefinery and circular economy.

Structure and function of negative-strand RNA virus polymerase complexes.

Viruses with negative-strand RNA genomes (NSVs) include many highly pathogenic and economically devastating disease-causing agents of humans, livestock, and plants-highlighted by recent Ebola and measles virus epidemics, and continuously circulating influenza virus. Because of their protein-coding orientation, NSVs face unique challenges for efficient gene expression and genome replication. To overcome these barriers, NSVs deliver a large and multifunctional RNA-dependent RNA polymerase into infected host cells. NSV-encoded polymerases contain all the enzymatic activities required for transcription and replication of their genome-including RNA synthesis and mRNA capping. Here, we review the structures and functions of NSV polymerases with a focus on key domains responsible for viral replication and gene expression. We highlight shared and unique features among polymerases of NSVs from the Mononegavirales, Bunyavirales, and Articulavirales orders.

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions.

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

The cap-specific m6A methyltransferase, PCIF1/CAPAM, is dynamically recruited to the gene promoter in a transcription-dependent manner.

N 6-methyladenosine (m6A), the most abundant modification in eukaryotic mRNAs, plays an important role in mRNA metabolism and functions. When adenosine is transcribed as the first cap-adjacent nucleotide, it is methylated at the ribose 2'-O and N6 positions, thus generating N6, 2'-O-dimethyladenosine (m6Am). Phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1) is a novel cap-specific adenine N6-methyltransferase responsible for m6Am formation. As PCIF1 specifically interacts with the Ser5-phosphorylated CTD of RNA polymerase II (Pol II), which is a marker for the early phase of transcription, PCIF1 is speculated to be recruited to the early elongating Pol II. In this study, subcellular fractionation and immunofluorescence microscopy demonstrated that PCIF1 is mainly localized to the transcriptionally active chromatin regions in HeLa cells. Chromatin immunoprecipitation (ChIP) revealed that PCIF1 was predominantly localized to the promoter of a broad range of Pol II-transcribed genes, including several protein-coding genes and non-coding RNA genes. Moreover, PCIF1 accumulation on these promoters depended entirely on transcriptional activity and Ser5 phosphorylation of the CTD. These results suggest that PCIF1 dynamically localizes to the Pol II early in transcription and may efficiently catalyze N6-methylation of the first adenosine residue of nascent mRNAs cotranscriptionally.

Targeting NSP16 Methyltransferase for the Broad-Spectrum Clinical Management of Coronaviruses: Managing the Next Pandemic.

With the approval and distribution of demonstrably safe COVID-19 vaccines bearing exceptionally high efficacy profiles, it may be tempting to envision a return to "normal" in the coming months. However, if there is one lesson to be learned from the ongoing pandemic, it is that, in a world of evolving zoonotic viruses, we must be better prepared for the next deadly outbreak. While the acute nature of the COVID-19 pandemic demanded a highly specific approach, it is advisable to consider the breadth of seemingly endless possibilities in our approach to managing the next inevitable occurrence of an outbreak. Though there is little chance of discovering a "magic pill" to combat all future pathogens, the highly conserved nature of non-surface viral proteins exposes an "Achilles' heel" in the structural genome of viral pathogens. Herein, we consider the potential of targeting such proteins to develop broad-spectrum therapeutics for the future. To illustrate this point, we outline the therapeutic potential of targeting the nonstructural protein 16 methyltransferase, which is conserved across most coronaviruses.

Evidence supporting a viral origin of the eukaryotic nucleus.

The defining feature of the eukaryotic cell is the possession of a nucleus that uncouples transcription from translation. According to the updated Viral Eukaryogenesis (VE) hypothesis presented here, the eukaryotic nucleus descends from the viral factory of a DNA virus that infected the archaeal ancestor of the eukaryotes. The VE hypothesis implies that many unique features of the nucleus, including the mechanisms by which the eukaryotic nucleus uncouples transcription from translation, should be viral rather than cellular in origin. The modern eukaryotic nucleus uncouples transcription from translation using a complex process employing hundreds of eukaryotic specific genes acting in concert. This intricate process is primed by the eukaryote specific 7-methylguanylate (m7G) cap on eukaryotic mRNA that targets mRNA for splicing, nuclear export, and cytoplasmic translation. It is shown here that homologues of the eukaryotic m7G capping apparatus are present in viruses of the Mimiviridae yet are apparently absent from archaea generally, and specifically from Lokiarchaeota, a proposed archaeal relative of the eukaryotes. Phylogenetic analysis of the m7G capping apparatus shows that eukaryotic nuclei and Mimiviridae obtained this shared pathway from a common ancestral source that predated the origin of the Last Eukaryotic Common Ancestor (LECA). These results are consistent with the hypothesis that the eukaryotic nucleus and the Mimiviridae obtained these abilities from an ancient virus that could be considered the 'First Eukaryotic Nuclear Ancestor' (FENA).

NELF Regulates a Promoter-Proximal Step Distinct from RNA Pol II Pause-Release.

RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.

Interplay of mRNA capping and transcription machineries.

Early stages of transcription from eukaryotic promoters include two principal events: the capping of newly synthesized mRNA and the transition of RNA polymerase II from the preinitiation complex to the productive elongation state. The capping checkpoint model implies that these events are tightly coupled, which is necessary for ensuring the proper capping of newly synthesized mRNA. Recent findings also show that the capping machinery has a wider effect on transcription and the entire gene expression process. The molecular basis of these phenomena is discussed.

RNA Synthesis and Capping by Non-segmented Negative Strand RNA Viral Polymerases: Lessons From a Prototypic Virus.

Non-segmented negative strand (NNS) RNA viruses belonging to the order Mononegavirales are highly diversified eukaryotic viruses including significant human pathogens, such as rabies, measles, Nipah, and Ebola. Elucidation of their unique strategies to replicate in eukaryotic cells is crucial to aid in developing anti-NNS RNA viral agents. Over the past 40 years, vesicular stomatitis virus (VSV), closely related to rabies virus, has served as a paradigm to study the fundamental molecular mechanisms of transcription and replication of NNS RNA viruses. These studies provided insights into how NNS RNA viruses synthesize 5'-capped mRNAs using their RNA-dependent RNA polymerase L proteins equipped with an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase (PRNTase), domain. PRNTase or PRNTase-like domains are evolutionally conserved among L proteins of all known NNS RNA viruses and their related viruses belonging to Jingchuvirales, a newly established order, in the class Monjiviricetes, suggesting that they may have evolved from a common ancestor that acquired the unique capping system to replicate in a primitive eukaryotic host. This article reviews what has been learned from biochemical and structural studies on the VSV RNA biosynthesis machinery, and then focuses on recent advances in our understanding of regulatory and catalytic roles of the PRNTase domain in RNA synthesis and capping.

Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein.

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5'-capping, cap methylation, and 3'-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay.

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses.

Cdk7: a kinase at the core of transcription and in the crosshairs of cancer drug discovery.

The transcription cycle of RNA polymerase II (Pol II) is regulated by a set of cyclin-dependent kinases (CDKs). Cdk7, associated with the transcription initiation factor TFIIH, is both an effector CDK that phosphorylates Pol II and other targets within the transcriptional machinery, and a CDK-activating kinase (CAK) for at least one other essential CDK involved in transcription. Recent studies have illuminated Cdk7 functions that are executed throughout the Pol II transcription cycle, from promoter clearance and promoter-proximal pausing, to co-transcriptional chromatin modification in gene bodies, to mRNA 3´-end formation and termination. Cdk7 has also emerged as a target of small-molecule inhibitors that show promise in the treatment of cancer and inflammation. The challenges now are to identify the relevant targets of Cdk7 at each step of the transcription cycle, and to understand how heightened dependence on an essential CDK emerges in cancer, and might be exploited therapeutically.

The large protein 'L' of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway.

Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus-Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein 'L' of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5'-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L-P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640-1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.