RESUMEN
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Biotinilación , Centrosoma/metabolismo , Simulación por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.
Asunto(s)
Calcineurina , Cilios , Calcineurina/metabolismo , Cilios/metabolismo , Calcio/metabolismo , Centrosoma/metabolismo , Centriolos/metabolismo , Proteínas/metabolismoRESUMEN
The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.
Asunto(s)
Proteínas de Ciclo Celular , Centrosoma , Puntos de Control de la Fase M del Ciclo Celular , Mitosis , Centrosoma/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/metabolismo , Huso Acromático/fisiología , División Celular , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Células HeLaRESUMEN
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
RESUMEN
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
RESUMEN
Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.
Asunto(s)
Axonema , Caenorhabditis elegans , Animales , Ratones , Axonema/metabolismo , Axonema/ultraestructura , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Mamíferos , Microtúbulos/metabolismo , Movimiento , Tubulina (Proteína)/metabolismoRESUMEN
The germline of Caenorhabditis elegans derives from a single founder cell, the germline blastomere P(4). P(4) is the product of four asymmetric cleavages that divide the zygote into distinct somatic and germline (P) lineages. P(4) inherits a specialized cytoplasm ("germ plasm") containing maternally encoded proteins and RNAs. The germ plasm has been hypothesized to specify germ cell fate, but the mechanisms involved remain unclear. Three processes stand out: (1) inhibition of mRNA transcription to prevent activation of somatic development, (2) translational regulation of the nanos homolog nos-2 and of other germ plasm mRNAs, and (3) establishment of a unique, partially repressive chromatin. Together, these processes ensure that the daughters of P(4), the primordial germ cells Z2 and Z3, gastrulate inside the embryo, associate with the somatic gonad, initiate the germline transcriptional program, and proliferate during larval development to generate â¼2,000 germ cells by adulthood.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Linaje de la Célula , Desarrollo Embrionario , Células Germinativas/citología , AnimalesRESUMEN
Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.
RESUMEN
Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50-100 µm from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 µm/minute. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium before other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.
Asunto(s)
Centriolos , Neuronas Receptoras Olfatorias , Animales , Centriolos/metabolismo , Cilios/metabolismo , Ratones , Microtúbulos , Mucosa OlfatoriaRESUMEN
Heart failure (HF) affects over 5.8 million patients in the United States, and can be very costly due to the number of hospitalizations and rehospitalizations during the final years of life. Due to the large number of hospitalizations for HF exacerbations, effective methods for preventing these occurrences are necessary. Improvements in the outpatient treatment of HF, aided by noninvasive and invasive home monitoring methods, can reduce the number of hospitalizations. Pulmonary pressure monitoring through the CardioMEMS system provides one method of hemodynamic assessment of patients. The efficacy of the CardioMEMS system in reducing the number of HF exacerbations has been explored in the CHAMPION trial (CardioMEMS Heart Sensor Allows Monitoring of Pressures to Improve Outcomes in NYHA Functional Class III Heart Failure Patients), which demonstrated a reduction in hospitalizations for HF exacerbations in patients whose medical management was guided by adjusting medications based on pulmonary pressures compared with clinical signs and symptoms. Retrospective analyses suggest that HF patients of certain subgroups, including those with left heart dysfunction and those with preserved left ventricular ejection fraction, could benefit from pulmonary pressure monitoring in controlling their HF. Larger studies are needed to determine whether mortality can be reduced with pulmonary pressure monitoring.
Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Monitoreo Fisiológico , Presión Esfenoidal Pulmonar/fisiología , Humanos , Función Ventricular Izquierda/fisiologíaRESUMEN
The centriole is a defining feature of many eukaryotic cells. It nucleates a cilium, organizes microtubules as part of the centrosome, and is duplicated in coordination with the cell cycle. Centrioles have a remarkable structure, consisting of microtubules arranged in a barrel with ninefold radial symmetry. At their base, or proximal end, centrioles have unique triplet microtubules, formed from three microtubules linked to each other. This microtubule organization is not found anywhere else in the cell, is conserved in all major branches of the eukaryotic tree, and likely was present in the last eukaryotic common ancestor. At their tip, or distal end, centrioles have doublet microtubules, which template the cilium. Here, we consider the structures of the compound microtubules in centrioles and discuss potential mechanisms for their formation and their function. We propose that triplet microtubules are required for the structural integrity of centrioles, allowing the centriole to serve as the essential nucleator of the cilium.
RESUMEN
Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.
Asunto(s)
Centriolos/metabolismo , Microtúbulos/metabolismo , Multimerización de Proteína , Tubulina (Proteína)/metabolismo , Eliminación de Gen , Humanos , Unión Proteica , Tubulina (Proteína)/genéticaRESUMEN
Screening for suppressor mutations is a powerful method to isolate genes that function in a common pathway or process. Because suppressor mutations often do not have phenotypes on their own, cloning of suppressor loci can be challenging. A method combining whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) mapping (WGS/SNP mapping) was developed to identify mutations with visible phenotypes in C. elegans. We show here that WGS/SNP mapping is an efficient method to map suppressor mutations without the need for previous phenotypic characterization. Using RNA-mediated interference to test candidate loci identified by WGS/SNP mapping, we identified 10 extragenic and six intragenic suppressors of mbk-2, a DYRK family kinase required for the transition from oocyte to zygote. Remarkably, seven suppressors are mutations in cell-cycle regulators that extend the timing of the oocyte-to-zygote transition.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Epistasis Genética , Genoma de los Helmintos , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Dominio Catalítico , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Análisis de Secuencia de ADNRESUMEN
RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2A(PPTR-½). Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/química , Proteína Fosfatasa 2/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN de Helminto/química , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Embrión no Mamífero , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Pliegue de Proteína , Proteína Fosfatasa 2/genética , Proteínas Tirosina Quinasas/genética , ARN de Helminto/genética , ARN de Helminto/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismoRESUMEN
Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.
Asunto(s)
Caenorhabditis elegans/embriología , Drosophila melanogaster/embriología , Embrión no Mamífero/ultraestructura , Imagenología Tridimensional/métodos , Microscopía/métodos , Imagen Molecular/métodos , Animales , Comunicación Celular , Células Madre Embrionarias/ultraestructura , Ratones , Esferoides Celulares/ultraestructuraRESUMEN
Lysine propionylation is a recently identified post-translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl-lysine is slightly reduced to acetyl-lysine. These results serve to broaden our understanding of the newly identified propionyl-lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.
Asunto(s)
Proteínas Bacterianas/química , Péptidos/química , Sirtuina 2/química , Acetilación , Cristalografía por Rayos X , Cinética , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Thermotoga maritima , Proteína p53 Supresora de Tumor/químicaRESUMEN
Asymmetric segregation of P granules during the first four divisions of the Caenorhabditis elegans embryo is a classic example of cytoplasmic partitioning of germline determinants. It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma. We have identified a mutant (pptr-1) in which P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres. Despite symmetric partitioning of P granule components, pptr-1 mutants segregate a germline that uniquely expresses P granules during postembryonic development. pptr-1 mutants are fertile, except at high temperatures. Hence, asymmetric partitioning of maternal P granules is not essential to specify germ cell fate. Instead, it may serve to protect the nascent germline from stress.
Asunto(s)
Blastómeros/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Citoplasma/metabolismo , Gránulos Citoplasmáticos/fisiología , Células Germinativas/fisiología , ARN de Helminto/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Gránulos Citoplasmáticos/ultraestructura , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Interfase , Microscopía Confocal , Mitosis , Mutación , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Cigoto/fisiologíaAsunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/genética , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ARN/genética , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.
Asunto(s)
Caperuzas de ARN , ARN Viral , Vesiculovirus/genética , Secuencia de Bases , Codón Iniciador , Guanosina Difosfato , Transcripción Genética , Estomatitis VesicularRESUMEN
Nonsegmented negative-sense (nsNS) RNA viruses typically replicate within the host cell cytoplasm and do not have access to the host mRNA capping machinery. These viruses have evolved a unique mechanism for mRNA cap formation in that the guanylyltransferase transfers GDP rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype nsNS RNA virus, we now provide genetic and biochemical evidence that its mRNA cap methylase activities are also unique. Using recombinant VSV, we determined the function in mRNA cap methylation of a predicted binding site in the polymerase for the methyl donor, S-adenosyl-l-methionine. We found that amino acid substitutions to this site disrupted methylation at the guanine-N-7 (G-N-7) position or at both the G-N-7 and ribose-2'-O (2'-O) positions of the mRNA cap. These studies provide genetic evidence that the two methylase activities share an S-adenosyl-l-methionine-binding site and show that, in contrast to other cap methylation reactions, methylation of the G-N-7 position is not required for 2'-O methylation. These findings suggest that VSV evolved an unusual strategy of mRNA cap methylation that may be shared by other nsNS RNA viruses.