The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.
- MeSH
- Antiviral Agents * pharmacology chemistry MeSH
- Fluorometry methods MeSH
- Humans MeSH
- Piperidines pharmacology MeSH
- Pyridines MeSH
- Pyrimidines MeSH
- Pyrroles MeSH
- RNA Caps metabolism MeSH
- RNA-Dependent RNA Polymerase antagonists & inhibitors metabolism MeSH
- High-Throughput Screening Assays * methods MeSH
- Viral Proteins * antagonists & inhibitors metabolism MeSH
- Influenza A virus drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.
- MeSH
- Genome, Viral MeSH
- Humans MeSH
- Transcription Initiation Site * MeSH
- Promoter Regions, Genetic MeSH
- RNA, Viral genetics MeSH
- Sequence Analysis, RNA * methods MeSH
- Gene Expression Profiling MeSH
- Transcriptome * MeSH
- Monkeypox virus genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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
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.
A carbon-carbon linkage is created when a methyl group is implanted on dUMP, thus resulting in the formation of dTMP by thymidylate synthase. The methyl group is deleted by aromatase when androgens are converted to estrogens. The methyl group is rearranged with the help of vitamin B12 in the isomerization of methylmalonyl-CoA to succinyl-CoA. S-adenosylmethionine (SAM) serves as the universal methyl donor involved in the biosynthesis of adrenaline and creatine(phosphate). It also interferes with the 5'-mRNA capping and the degradation of catecholamines (i.e. adrenaline, noradrenaline). Cholesterol could be viewed as a conglomeration of methyl groups. Finally, as part of valine, two methyl functions participate in the origin of one of the most frequent hereditary diseases on earth, sickle cell anemia.
- MeSH
- Epinephrine MeSH
- Cholesterol * MeSH
- Vitamin B 12 * metabolism MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.
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
Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.
- MeSH
- eIF-2 Kinase genetics metabolism MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Mice MeSH
- Tumor Suppressor Protein p53 * genetics metabolism MeSH
- Protein Processing, Post-Translational MeSH
- Protein Isoforms metabolism MeSH
- Endoplasmic Reticulum Stress * genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.
- MeSH
- Cell Nucleolus genetics metabolism MeSH
- DEAD-box RNA Helicases metabolism MeSH
- Nuclear Proteins genetics metabolism MeSH
- Humans MeSH
- Cell Adhesion Molecules metabolism MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 genetics metabolism MeSH
- RNA Precursors metabolism MeSH
- Ribosomal Proteins * genetics metabolism MeSH
- Ribosomes genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.
- MeSH
- Transcription, Genetic genetics MeSH
- Hypertension * genetics metabolism MeSH
- Rats MeSH
- Rats, Inbred SHR MeSH
- Promoter Regions, Genetic genetics MeSH
- Sequence Analysis, RNA methods MeSH
- Gene Expression Profiling methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
