Cap Structure Research Articles

Production of Fully Capped mRNA for Transfection into Mammalian Cells: A Protocol for Enzymatic Degradation of Uncapped Transcripts After In Vitro mRNA Synthesis.

The functionality of messenger RNA, such as stability and translation, is determined by several elements. In Eukaryotes, the 5' end of the mRNA is modified to contain a 5' cap structure, the presence of which protects the mRNA from degradation by 5' to 3' exoribonucleases and promotes mRNA translation. The in vitro synthesis of RNA has recently attracted ample attention for its application as a source of therapeutic agents or research tools. Although in vitro mRNA synthesis (IVT) methodology is well established and is still being improved, not all synthesized RNA molecules have the expected properties. For example, the co-transcriptional addition of a 5' cap is frequently incomplete. Yet, the uncapped mRNA molecules are undesirable and must be removed before further processing. Here, we present a protocol for the enzymatic removal of uncapped RNA molecules. This approach offers an excellent opportunity to enrich IVT products with fully competent, 5' cap-containing mRNA molecules.

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Experimental Study of Wave Load Distributions on Pile Groups Affected by Cap Structures and Pile Spacings Under Varied Wave Conditions

Experimental Study of Wave Load Distributions on Pile Groups Affected by Cap Structures and Pile Spacings Under Varied Wave Conditions

The Cap and the Spermatostyle Protecting the Sperm Bundle Have a Similar Origin-Ultrastructural Study of the Spermatogenesis from the Ground Beetle Carabus (Chaetocarabus) lefebvrei Dejean, 1826 (Adephaga Carabidae).

The males of Carabus lefebvrei produce sperm bundles of quite variable size as a consequence of a sexual selection via sperm competition. The ultrastructural features of spermatogenesis of the species and the sequence of events leading to sperm bundle formation along the deferent duct lumen revealed the origin and evolution of the apical structure protecting the sperm bundles. Elongated layers of the electron-dense material of variable size and thickness are visible over the microvilli of the distal deferent duct epithelium. They likely correspond to sections through different levels of discoidal secretions. Then, the sperm present in the duct lumen attach to one side of such elongated secreted structures. Later on, these structures bent to take a cap shape and increase their size and thickness by apposition of additional secretions produced by the proximal deferent duct epithelium. These results reconcile the different opinions about the formation of the structures called the cap and the rod (spermatostyle). In our work, we in fact obtained evidence that both these structures have the same origin, even though at the end of their formation they exhibit a different appearance. The reported results indicate that, differently from the spermatostyle, in the initial stages of cap formation, the sperm adhere only to one side of the cap rather than around the whole structure, as it occurs instead along the spermatostyle rod. Under this aspect, the cap structure is similar to the protection present in the sperm of some whirligig beetle.

Human DCP1 is crucial for mRNA decapping and possesses paralog-specific gene regulating functions.

The mRNA 5'-cap structure removal by the decapping enzyme DCP2 is a critical step in gene regulation. While DCP2 is the catalytic subunit in the decapping complex, its activity is strongly enhanced by multiple factors, particularly DCP1, which is the major activator in yeast. However, the precise role of DCP1 in metazoans has yet to be fully elucidated. Moreover, in humans, the specific biological functions of the two DCP1 paralogs, DCP1a and DCP1b, remain largely unknown. To investigate the role of human DCP1, we generated cell lines that were deficient in DCP1a, DCP1b, or both to evaluate the importance of DCP1 in the decapping machinery. Our results highlight the importance of human DCP1 in decapping process and show that the EVH1 domain of DCP1 enhances the mRNA-binding affinity of DCP2. Transcriptome and metabolome analyses outline the distinct functions of DCP1a and DCP1b in human cells, regulating specific endogenous mRNA targets and biological processes. Overall, our findings provide insights into the molecular mechanism of human DCP1 in mRNA decapping and shed light on the distinct functions of its paralogs.

M6Am Methyltransferase PCIF1 Promotes LPP3 Mediated Phosphatidic Acid Metabolism and Renal Cell Carcinoma Progression.

N6-methyl-2'-O-methyladenosine (m6Am), occurring adjacent to the 7-methylguanosine (m7G) cap structure and catalyzed by the newly identified writer PCIF1 (phosphorylated CTD interacting factor 1), has been implicated in the pathogenesis of various diseases. However, its involvement in renal cell carcinoma (RCC) remains unexplored. Here, significant upregulation of PCIF1 and m6Am levels in RCC tissues are identified, unveiling their oncogenic roles both in vitro and in vivo. Mechanically, employing m6Am-Exo-Seq, LPP3 (phospholipid phosphatase 3) mRNA is identified as a key downstream target whose translation is enhanced by m6Am modification. Furthermore, LPP3 is revealed as a key regulator of phosphatidic acid metabolism, critical for preventing its accumulation in mitochondria and facilitating mitochondrial fission. Consequently, Inhibition of the PCIF1/LPP3 axis significantly altered mitochondrial morphology and reduced RCC tumor progression. In addition, depletion of PCIF1 sensitizes RCC to sunitinib treatment. This study highlights the intricate interplay between m6Am modification, phosphatidic acid metabolism, and mitochondrial dynamics, offering a promising therapeutic avenue for RCC.

2'-O-methyltransferase-deficient yellow fever virus: Restricted replication in the midgut and secondary tissues of Aedes aegypti mosquitoes severely limits dissemination.

The RNA genome of orthoflaviviruses encodes a methyltransferase within the non-structural protein NS5, which is involved in 2'-O-methylation of the 5'-terminal nucleotide of the viral genome resulting in a cap1 structure. While a 2'-O-unmethylated cap0 structure is recognized in vertebrates by the RNA sensor RIG-I, the cap1 structure allows orthoflaviviruses to evade the vertebrate innate immune system. Here, we analyzed whether the cap0 structure is also recognized in mosquitoes. Replication analyses of 2'-O-methyltransferase deficient yellow fever virus mutants (YFV NS5-E218A) of the vaccine 17D and the wild-type Asibi strain in mosquito cells revealed a distinct downregulation of the cap0 viruses. Interestingly, the level of inhibition differed for various mosquito cells. The most striking difference was found in Aedes albopictus-derived C6/36 cells with YFV-17D cap0 replication being completely blocked. Replication of YFV-Asibi cap0 was also suppressed in mosquito cells but to a lower extent. Analyses using chimeras between YFV-17D and YFV-Asibi suggest that a synergistic effect of several mutations across the viral genome accompanied by a faster initial growth rate of YFV-Asibi cap1 correlates with the lower level of YFV-Asibi cap0 attenuation. Viral growth analyses in Dicer-2 knockout cells demonstrated that Dicer-2 is entirely dispensable for attenuating the YFV cap0 viruses. Translation of a replication-incompetent cap0 reporter YFV-17D genome was reduced in mosquito cells, indicating a cap0 sensing translation regulation mechanism. Further, oral infection of Aedes aegypti mosquitoes resulted in lower infection rates for YFV-Asibi cap0. The latter is related to lower viral loads found in the midguts, which largely diminished dissemination to secondary tissues. After intrathoracic infection, YFV-Asibi cap0 replicated slower and to decreased amounts in secondary tissues compared to YFV-Asibi cap1. These results suggest the existence of an ubiquitously expressed innate antiviral protein recognizing 5'-terminal RNA cap-modifications in mosquitoes, both in the midgut as well as in secondary tissues.

Design and analysis of hundred-meter wind turbine blade trailing edge based on finite element method (FEM)

Abstract The trailing edge region of composite wind turbine blades usually has a large deformation under wind load and the accidents caused by the damage of blade trailing edge accounts for a large proportion among blade accidents. With the development of large blade size, higher requirements are put forward for the stability of wind turbine blade trailing edge. Therefore, the trailing edge region is critical in the design of blade. A detailed study of the design and analysis for wind turbine blade trailing edge region was carried out in which the structure stability was regarded as the key. The influence of auxiliary spar cap and auxiliary spar web on the stiffness distribution and stability of the trailing edge were studied based on focus and finite element method (FEM). The respiratory effect of different structures due to deformation were analyzed and compared. The results showed that the structure of the auxiliary spar cap and auxiliary spar web can significantly increase stiffness, stability and decrease respiratory deformation at the trailing edge of blade compared to the traditional three webs structure. The structure of auxiliary spar and auxiliary spar web is the best design scheme for hundred-meter composite blade trailing edge.

The expanding role of cap-adjacent modifications in animals

Eukaryotic mRNA cap structures directly influence mRNA stability, translation, and immune recognition. While the significance of the mRNA cap itself has been well-established, recent research has revealed the intricate modifications to the nucleotides immediately adjacent to the cap. These include 2′-O-methylation (Cap1, Cap2) and N6-methyladenosine (m6Am), which recent work suggests may be dynamically regulated, interdependent, and vital for gene-regulation. This perspective explores the expanding role of cap-adjacent modifications. Cap1 is found on metazoan mRNA, viruses which infect metazoans, and the COVID-19 mRNA vaccines. We combined basic local alignment and 2D-TLC to track cap-adjacent modifying proteins and the modifications themselves to profile their prevalence throughout eukaryotes. We confirm that Cap1 is broadly metazoan specific, but surprisingly also in a brown algae. Additionally, we find genomic presence of the eraser, FTO more predictive of the occurrence of m6Am than the m6Am-writer, PCIF1. We then outline future directions to understand and exploit these enigmatic modifications.

Fibre Refractometry for Minimally Invasive Sugar Content Measurements within Produce

A minimally invasive needle refractometer is presented for sugar content measurements within produce. A passive sampling cap structure was developed that improves the reliability of the device by avoiding interfering back reflections from the flesh of the produce. It is explained that factory calibration may not be needed for this type of refractometer, potentially reducing production costs. Also demonstrated is an iterative method to correct for temperature variations without the need for an integrated model for how the refractive index changes with temperature for different levels of sugar concentration. The sensor showed a typical standard deviation of 0.4 °Bx for a 10-s-long measurement and was validated against a prism refractometer, showing an average offset of (0.0±0.1) °Bx. In addition, the potential for using the device to investigate sugar distributions within a single fruit sample is demonstrated.

Synthesis and anti-influenza virus activity of substituted dibenzoxepine-based baloxavir derivatives

Seasonal influenza poses a significant threat to global public health, driving the need for effective anti-influenza agents. The PA protein, which captures the pre-mRNA cap structure, is crucial for the replication of the influenza virus and serves as an important target for developing such agents. Baloxavir, a PA inhibitor, has shown excellent activity against influenza A and B viruses. In this study, its structure was optimized using bioisosteric replacement to develop novel dibenzoxepine-based derivatives for combating influenza. As the lead compounds, ATV03 (EC50 = 0.78 ± 0.10 nM, SI > 64103) and ATV07 (EC50 = 0.78 ± 0.01 nM, SI = 31603) demonstrated excellent anti-influenza A (H3N2) activity and SI, and possessed favorable anti-influenza B activity, with 2.02 ± 0.40 nM and 2.32 ± 0.29 nM of EC50 respectively. They showed improved bioavailability and metabolic stability. Mechanism studies revealed that ATV03 and ATV07 both possessed significant activity in inhibiting PA and RdRp as well as disturbing NP. Consequently, ATV03 was selected for further investigation in the fight against seasonal and pandemic influenza due to its superior bioavailability, metabolic stability, and efficacy against multiple influenza A viruses.

The P(III)-Amidite Based Synthesis of Stable Isotope Labeled mRNA-Cap-Structures Enables their Sensitive Quantitation from Brain Tissue.

The 5' cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high-quality synthetic standards via a new route, involving P(III)-amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one-step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl-alcohols. Stable-isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC-MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences.

Application of Mammalian Nudix Enzymes to Capped RNA Analysis.

Following the success of mRNA vaccines against COVID-19, mRNA-based therapeutics have now become a great interest and potential. The development of this approach has been preceded by studies of modifications found on mRNA ribonucleotides that influence the stability, translation and immunogenicity of this molecule. The 5' cap of eukaryotic mRNA plays a critical role in these cellular functions and is thus the focus of intensive chemical modifications to affect the biological properties of in vitro-prepared mRNA. Enzymatic removal of the 5' cap affects the stability of mRNA in vivo. The NUDIX hydrolase Dcp2 was identified as the first eukaryotic decapping enzyme and is routinely used to analyse the synthetic cap at the 5' end of RNA. Here we highlight three additional NUDIX enzymes with known decapping activity, namely Nudt2, Nudt12 and Nudt16. These enzymes possess a different and some overlapping activity towards numerous 5' RNA cap structures, including non-canonical and chemically modified ones. Therefore, they appear as potent tools for comprehensive in vitro characterisation of capped RNA transcripts, with special focus on synthetic RNAs with therapeutic activity.

Deciphering EIF3D's Role in Immune Regulation and Malignant Progression: A Pan-Cancer Analysis with a Focus on Colon Adenocarcinoma.

EIF3D, a key component of the eukaryotic translation initiation factor 3 (EIF3) complex, is critical in selectively translating mRNAs with atypical cap structures. Its relationship with colon adenocarcinoma (COAD) development and immune infiltration, however, remains under-explored. This study delves into EIF3D's role in COAD using bioinformatics and in vitro experimentation. We analyzed EIF3D expression levels utilizing TCGA, GTEx, CPTAC, and TISIDB databases. The TISCH database and ssGSEA method helped in assessing EIF3D's link with the tumor immune microenvironment. EIF3D expression in CRC cells was gauged via real-time PCR. Cell proliferation was assessed using CCK8 and colony formation assays, while migration capabilities were tested through Transwell assays. Flow cytometry facilitated cell cycle distribution and apoptosis analysis. ChIP-qPCR identified transcription factors regulating EIF3D, and bulk sequencing explored EIF3D's pathways in promoting COAD. EIF3D upregulation is a common feature in various tumors, especially in COAD, correlating with poor prognosis in many cancer types. It showed significant associations with immune cell and cancer-associated fibroblast (CAF) infiltration across multiple tumors. Additionally, it is closely associated with molecular and immune subtypes of multiple tumors, including COAD. Single-cell analyses depicted EIF3D's distribution and proportion in CRC immune cells. In vitro findings indicated EIF3D knockdown curtailed proliferation and migration, inducing G0/G1 arrest in COAD cells. Moreover, bulk sequencing revealed EIF3D knockdown interferes with multiple cancer-related pathways, likely by curtailing cell cycle and DNA replication activities to regulate cell proliferation. EIF3D emerges as a potential prognostic biomarker for tumor progression and immune infiltration, particularly in COAD, potentially predicting immunotherapy efficacy. Additionally, EIF3D represents a multifaceted target implicated in COAD's malignant progression.

M7GRegpred: substrate prediction of N7-methylguanosine (m7G) writers and readers based on sequencing features.

N7-Methylguanosine (m7G) is important RNA modification at internal and the cap structure of five terminal end of message RNA. It is essential for RNA stability of RNA, the efficiency of translation, and various intracellular RNA processing pathways. Given the significance of the m7G modification, numerous studies have been conducted to predict m7G sites. To further elucidate the regulatory mechanisms surrounding m7G, we introduce a novel bioinformatics framework, m7GRegpred, designed to forecast the targets of the m7G methyltransferases METTL1 and WDR4, and m7G readers QKI5, QKI6, and QKI7 for the first time. We integrated different features to build predictors, with AUROC scores of 0.856, 0.857, 0.780, 0.776, 0.818 for METTL1, WDR4, QKI5, QKI6, and QKI7, respectively. In addition, the effect of window lengths and algorism were systemically evaluated in this work. The finial model was summarized in a user-friendly webserver: http://modinfor.com/m7GRegpred/. Our research indicates that the substrates of m7G regulators can be identified and may potentially advance the study of m7G regulators under unique conditions.

Identification of Two Long Noncoding RNAs, Kcnq1ot1 and Rmst, as Biomarkers in Chronic Liver Diseases in Mice.

This study investigates novel short-lived long noncoding RNAs (lncRNAs) in mice with altered expression in metabolic dysfunction-associated steatotic liver (MASH) and liver fibrosis. LncRNAs share similarities with mRNAs in their transcription by RNA polymerase II, possession of a 5' cap structure, and presence of a polyA tail. We identified two lncRNAs, Kcnq1ot1 and Rmst, significantly decreased in both conditions. These lncRNAs showed dramatic expression changes in MASH livers induced by Western diets and CCl4, and in fibrotic livers induced by CCl4 alone. The decrease was more pronounced in liver fibrosis, suggesting their potential as biomarkers for disease progression. Our findings are consistent across different fibrosis models, indicating a crucial role for these lncRNAs in MASH and liver fibrosis in mice. With MASH becoming a global health issue and its progression to fibrosis associated with hepatocarcinogenesis and poor prognosis, understanding the underlying mechanisms is critical. This research contributes to elucidating lncRNA functions in murine liver diseases and provides a foundation for developing novel therapeutic strategies targeting lncRNAs in MASH and liver fibrosis, offering new avenues for potential therapeutic interventions.

Respiratory Syncytial Virus (RSV) optimizes the translational landscape during infection.

Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5'-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5'-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

Targeting Monkeypox Virus Methyltransferase: Virtual Screening of Natural Compounds from Middle-Eastern Medicinal Plants.

Monkeypox is an infectious disease resulting from the monkeypox virus, and its fatality rate varies depending on the virus clade and the location of the outbreak. In monkeypox virus, methyltransferase (MTase) plays a crucial role in modifying the cap structure of viral mRNA. This alteration assists the virus in evading the host's immune system, enhances viral protein synthesis, and ultimately enables successful infection and replication within host cells. Given the significance of MTase in viral infection and spread within the host, our study aimed to identify a natural inhibitor for this enzyme using docking and molecular dynamic (MD) simulations. We collected a total of 12,971 natural compounds from 200 medicinal plants in the Middle East. After eliminating duplicate compounds, we had 5,749 unique ligand conformers, which we then subjected to high-throughput virtual screening against MTase. The most promising hits were further evaluated using the extra-precision (XP) tool. The affinity of these hits was also assessed by Prime-Molecular Mechanics/Generalized Born Surface Area (MMGBSA) tool. The analysis revealed that two standard controls (sinefungin and TO1119) and two Middle-Eastern compounds (folic acid and 1,2,4,6-tetragalloylglucose) exhibited the best XP docking scores. According to Prime MMGBSA calculations, the Middle-Eastern compounds showed higher affinities, with values of -60.61 kcal/mol for 1,2,4,6-tetragalloylglucose and -51.87 kcal/mol for folic acid, surpassing the controls (TO1119 at -35.71 kcal/mol and sinefungin at -31.51 kcal/mol). In the majority of Molecular dynamic (MD) simulations, folic acid exhibited demonstrated greater stability than sinefungin. Further investigation revealed that folic acid occupied a critical position in the active site of MTase, which reduced its interaction with the mRNA substrate. Based on these findings, it can be concluded that folic acid is a highly promising natural compound for potential use in the cost-effective treatment of monkeypox virus. The identification of folic acid as a potential antiviral agent highlights the importance of nature in providing new therapeutic uses that have significant implications for global health, particularly in regions where monkeypox viral outbreaks are prevalent. However, it is essential to note that further wet-lab validations are necessary to confirm its efficacy for treatment in a medical context.

The complete pathway for co-transcriptional mRNA maturation within a large protein of a non-segmented negative-strand RNA virus.

Non-segmented negative-strand (NNS) RNA viruses, such as rabies, Nipahand Ebola, produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs. Here, we developed a transcription elongation-coupled pre-mRNA capping system for vesicular stomatitis virus (VSV, a prototypic NNS RNA virus). Using this system, we demonstrate that the single-polypeptide RNA-dependent RNA polymerase (RdRp) large protein (L) catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N7-methylation of GpppAm into m7GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. The GDP polyribonucleotidyltransferase (PRNTase) domain of L generated capped pre-mRNAs of 18 nucleotides or longer via the formation of covalent enzyme-pre-mRNA intermediates. The single methyltransferase domain of L sequentially methylated the cap structure only when pre-mRNAs of 40 nucleotides or longer were associated with elongation complexes. These results suggest that the formation of pre-mRNA closed loop structures in elongation complexes via the RdRp and PRNTase domains followed by the RdRp and MTase domains on the same polypeptide is required for the cap 1 formation during transcription. Taken together, our findings indicate that NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery.

Validation of a questionnaire for Central nervous system Aspects of joint Pain: the CAP questionnaire.

Neuropathic-like pain, fatigue, cognitive difficulty, catastrophising, anxiety, sleep disturbance, depression, and widespread pain associate with a single factor in people with knee pain. We report the Central Aspects of Pain questionnaire (CAP) to characterise this across painful musculoskeletal conditions. CAP was derived from the 8 item CAP-Knee questionnaire, and completed by participants with joint pain in the Investigating Musculoskeletal Health and Wellbeing survey. Subgroups had osteoarthritis, back pain or fibromyalgia. Acceptability was evaluated by feedback and data missingness. Correlation coefficients informed widespread pain scoring threshold in relation to the other items, and evaluated associations with pain. Factor analysis assessed CAP structure. Intraclass Correlation Coefficient (ICC) between paper and electronic administration assessed reliability. Friedman test assessed score stability over 4 years in people reporting knee osteoarthritis. Data were from 3579 participants (58% female, median age; 71 years), including subgroups with osteoarthritis (n = 1158), back pain (n = 1292) or fibromyalgia (n = 177). Across the 3 subgroups, ≥10/26 painful sites on the manikin scored widespread pain. Reliability was high (ICC= 0.89 (95% CI: 0.84-0.92)) and CAP scores fit to 1 and 2 factor model, with a total CAP score that was associated with pain severity and quality (r = 0.50-0.72). In people with knee pain, CAP scores were stable over 4 years at the group level, but displayed significant temporal heterogeneity within individual participants. Central Aspects of Pain is reliably measured by the CAP questionnaire across a range of painful musculoskeletal conditions, and is a changeable state.