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2.
BMC Bioinformatics ; 23(Suppl 3): 559, 2022 Dec 23.
Article in English | MEDLINE | ID: mdl-36564729

ABSTRACT

BACKGROUND: RNA secondary structure has broad impact on the fate of RNA metabolism. The reduced stability of secondary structures near the translation initiation site/start codon of the coding region promotes the efficiency of translation in both prokaryotic and eukaryotic species. However, the inaccuracy of in silico folding and the focus on the coding region limit our understanding of the global relationship between the whole mRNA structure and translation efficiency. Leveraging high-throughput RNA structure probing data in the transcriptome, we aim to systematically investigate the role of RNA structure in regulating translation efficiency. RESULTS: Here, we analyze the influences of hundreds of sequence and structural features on translation efficiency in the mouse embryonic stem cells (mESCs) and zebrafish developmental stages. Our findings reveal that overall in vivo RNA structure has a higher relative importance in predicting translation efficiency than in vitro RNA structure in both mESCs and zebrafish. Also, RNA structures in 3' untranslated region (UTR) have much stronger influence on translation efficiency compared to those in coding regions or 5' UTR. Furthermore, strong alternation between in vitro and in vivo structures in 3' UTR are detected in highly translated mRNAs in mESCs but not zebrafish. Instead, moderate alteration between in vitro and in vivo RNA structures in the 5' UTR and proximal coding regions are detected in highly translated mRNAs in zebrafish. CONCLUSIONS: Our results suggest the openness of the 3' UTR promotes the translation efficiency in both mice and zebrafish, with the in vivo structure in 3' UTR more important in mice than in zebrafish. This reveals a novel role of RNA secondary structure on translational regulation.


Subject(s)
Eukaryotic Cells , Protein Biosynthesis , Animals , Mice , 5' Untranslated Regions , 3' Untranslated Regions , RNA, Messenger/genetics , RNA, Messenger/chemistry
3.
ACS Appl Mater Interfaces ; 14(51): 56440-56453, 2022 Dec 28.
Article in English | MEDLINE | ID: mdl-36525379

ABSTRACT

Extrahepatic nucleic acid delivery using polymers typically requires the synthesis and purification of custom monomers, post-synthetic modifications, and incorporation of additional excipients to augment their stability, endosomal escape, and in vivo effectiveness. Here, we report the development of a single-component and excipient-free, polyester-based nucleic acid delivery nanoparticle platform comprising ionizable N-methyldiethanolamine (MDET) and various hydrophobic alkyl diols (Cp) that achieves lung-selective nucleic acid transfection in vivo. PolyMDET and polyMDET-Cp polyplexes displayed high serum and enzymatic stability, while delivering pDNA or mRNA to "hard-to-transfect" innate immune cells. PolyMDET-C4 and polyMDET-C6 mediated high protein expression in lung alveolar macrophages and dendritic cells without inducing tissue damage or systemic inflammatory responses. Improved strategies using readily available starting materials to produce a simple, excipient-free, non-viral nucleic acid delivery platform with lung-selective and innate immune cell tropism has the potential to expedite clinical deployment of polymer-based genetic medicines.


Subject(s)
Nanoparticles , Polyesters , RNA, Messenger/genetics , RNA, Messenger/chemistry , Transfection , Plasmids/genetics , DNA/chemistry , Polymers/chemistry , Lung/metabolism , Nanoparticles/chemistry , Immunity, Innate
4.
Nano Lett ; 22(24): 10025-10033, 2022 12 28.
Article in English | MEDLINE | ID: mdl-36521071

ABSTRACT

Lipid nanoparticles (LNPs) have delivered therapeutic RNA to hepatocytes in humans. Adsorption of apolipoprotein E (ApoE) onto these clinical LNP-mRNA drugs has been shown to facilitate hepatocyte entry via the low-density lipoprotein receptor (LDLR). Since ApoE-LDLR trafficking is conserved in mice, non-human primates, and humans, characterizing this mechanism eased clinical transition. Recently, LNPs have delivered mRNA to non-hepatocytes in mice and non-human primates, suggesting they can target new cell types via ApoE- and LDLR-independent pathways. To test this hypothesis, we quantified how 60 LNPs delivered mRNA with cell type resolution in wild-type mice and three knockout mouse strains related to lipid trafficking: ApoE-/-, LDLR-/-, and PCSK9-/-. These data suggest that the hydrophobic tail length of diketopiperazine-based lipids can be changed to drive ApoE- and LDLR-independent delivery in vivo. More broadly, the results support the hypothesis that endogenous LNP trafficking can be tuned by modifying lipid chemistry.


Subject(s)
Apolipoproteins E , Lipoproteins, LDL , Nanoparticles , Animals , Mice , Apolipoproteins E/genetics , Lipoproteins, LDL/genetics , Mice, Knockout , Nanoparticles/chemistry , RNA, Messenger/chemistry
5.
Proc Natl Acad Sci U S A ; 119(44): e2212502119, 2022 11.
Article in English | MEDLINE | ID: mdl-36282914

ABSTRACT

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.


Subject(s)
Peptide Elongation Factor G , Ribosomes , Peptide Elongation Factor G/genetics , Peptide Elongation Factor G/chemistry , Guanosine Triphosphate/chemistry , Hydrolysis , Ribosomes/metabolism , RNA, Transfer/chemistry , RNA, Messenger/chemistry , GTP Phosphohydrolases/genetics , Pyrenes/analysis , Guanosine
6.
ACS Nano ; 16(11): 18936-18950, 2022 Nov 22.
Article in English | MEDLINE | ID: mdl-36269150

ABSTRACT

Ionizable cationic lipid-containing lipid nanoparticles (LNPs) are the most clinically advanced non-viral gene delivery platforms, holding great potential for gene therapeutics. This is exemplified by the two COVID-19 vaccines employing mRNA-LNP technology from Pfizer/BioNTech and Moderna. Herein, we develop a chemical library of ionizable cationic lipids through a one-step chemical-biological enzyme-catalyzed esterification method, and the synthesized ionizable lipids were further prepared to be LNPs for mRNA delivery. Through orthogonal design of experiment methodology screening, the top-performing AA3-DLin LNPs show outstanding mRNA delivery efficacy and long-term storage capability. Furthermore, the AA3-DLin LNP COVID-19 vaccines encapsulating SARS-CoV-2 spike mRNAs successfully induced strong immunogenicity in a BALB/c mouse model demonstrated by the antibody titers, virus challenge, and T cell immune response studies. The developed AA3-DLin LNPs are an excellent mRNA delivery platform, and this study provides an overall perspective of the ionizable cationic lipids, from aspects of lipid design, synthesis, screening, optimization, fabrication, characterization, and application.


Subject(s)
COVID-19 , Nanoparticles , Mice , Animals , Humans , RNA, Messenger/genetics , RNA, Messenger/chemistry , COVID-19 Vaccines , Lipids/chemistry , COVID-19/prevention & control , SARS-CoV-2/genetics , Nanoparticles/chemistry , Liposomes , Cations , Catalysis
7.
J Transl Med ; 20(1): 476, 2022 10 20.
Article in English | MEDLINE | ID: mdl-36266694

ABSTRACT

RNA methylation modifications, especially m6A mRNA modification, are known to be extensively involved in tumor development. However, the relationship between N3-methylcytidine (m3C) related genes and tumorigenesis has rarely been studied. In this research, we found that m3C-related genes were expressed at different levels and affected patients' prognosis across multiple cancer types from The Cancer Genome Atlas and multi-omics levels. Importantly, methyltransferase-like proteins 2A (METTL2A) had a high amplification frequency (~ 7%) in patients with breast invasive carcinoma (BRCA), and its overexpression was an independent predictor of poor overall survival. Enrichment analysis of associated genes revealed that METTL2A may activate DNA synthesis and cell proliferation pathways in BRCA cells. Through drug sensitivity analysis, Trifluridine, PD407824, and Taselisib were shown to be effective drugs for METTL2A-positive BRCA patients. Overall, our research conducts a holistic view of the expression level and prognostic signature of m3C-related genes with multiple malignancies. Importantly, METTL2A has been intensely explored as a potential oncogene in BRCA, to aid the development of potential drug agents for precision therapy in breast cancer patients.


Subject(s)
Breast Neoplasms , tRNA Methyltransferases , Female , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA , Oncogenes/genetics , RNA , RNA, Messenger/chemistry , Trifluridine , tRNA Methyltransferases/genetics
8.
Nat Commun ; 13(1): 5561, 2022 09 23.
Article in English | MEDLINE | ID: mdl-36151112

ABSTRACT

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%-80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.


Subject(s)
Lipids , Nanoparticles , Lipids/chemistry , Liposomes , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Small Interfering/genetics
9.
Front Biosci (Elite Ed) ; 14(3): 17, 2022 07 04.
Article in English | MEDLINE | ID: mdl-36137989

ABSTRACT

BACKGROUND: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. METHODS: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. RESULTS: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. CONCLUSIONS: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.


Subject(s)
Eukaryotic Initiation Factor-4F , Eukaryotic Initiation Factor-4G , Eukaryotic Initiation Factor-4F/genetics , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Ferritins/genetics , Iron/metabolism , Iron-Regulatory Proteins/genetics , Iron-Regulatory Proteins/metabolism , RNA Caps/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Untranslated Regions
10.
Mol Pharm ; 19(11): 4275-4285, 2022 11 07.
Article in English | MEDLINE | ID: mdl-36129254

ABSTRACT

Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers─HEPES, Tris, and phosphate-buffered saline─with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze-thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.


Subject(s)
COVID-19 , Nanoparticles , Humans , RNA, Messenger/genetics , RNA, Messenger/chemistry , Lipids/chemistry , Pandemics , Nanoparticles/chemistry , Liposomes , RNA, Small Interfering/chemistry
11.
Int J Mol Sci ; 23(17)2022 Aug 26.
Article in English | MEDLINE | ID: mdl-36077109

ABSTRACT

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA's structure and its interactions with proteins in the translation process. In particular, the 5'-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5'-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5'-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5'-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.


Subject(s)
RNA, Messenger/chemistry , Tumor Suppressor Protein p53/genetics , Animals , Mice , Nucleic Acid Hybridization , RNA, Messenger/metabolism , Scattering, Small Angle , Tumor Suppressor Protein p53/metabolism , X-Ray Diffraction
12.
Int J Mol Sci ; 23(17)2022 Aug 28.
Article in English | MEDLINE | ID: mdl-36077143

ABSTRACT

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.


Subject(s)
Methyltransferases , RNA, Messenger/metabolism , HEK293 Cells , Humans , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , RNA, Messenger/chemistry
13.
J Mol Biol ; 434(20): 167801, 2022 10 30.
Article in English | MEDLINE | ID: mdl-36038000

ABSTRACT

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA's are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.


Subject(s)
Neurons , Protein Biosynthesis , RNA, Messenger , Ribosomes , Stress Granules , Animals , Cryoelectron Microscopy , Eukaryotic Initiation Factor-3/genetics , Humans , Neurons/metabolism , Neurons/ultrastructure , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Rats , Ribosome Subunits, Small, Eukaryotic , Ribosomes/metabolism , Stress Granules/chemistry
14.
Biomaterials ; 288: 121738, 2022 09.
Article in English | MEDLINE | ID: mdl-36008186

ABSTRACT

Despite DNA nanotechnology has spawned a broad variety and taken a giant leap toward cancer theranostic applications over the last decade, the homogeneous DNA nanostructures often suffer from fatal degradation due to their limited stability and specificity. Herein, for the first time, we report a stable DNA tetrahedra-gold nanoclusters (DT/AuNCs) nanohybrid with a self-assembly/programmed disassembly manner for stimuli-responsive tumor imaging and gene-chemo therapy. By utilizing the multifunctional peptides with positive and legumain-specific domains as bioligands, AuNCs were synthesized as signal generators and gate guard attached on the dual-responsive DT, forming the DT/AuNCs with sequential response to legumain-TK1 mRNA & glutathione. The tumorous biomarker of legumain initiated the signal generation relying on the nanosurface energy transfer effect of AuNCs and denudation of DT-Dox (preliminary disassembly). Successively, the dual-responsive DT-Dox administrated a sequential fragmentation along with Dox release in response to the up-regulated glutathione and TK1 mRNA (secondary disassembly), thereby leading to combined gene silencing and chemo-therapy. The results revealed that the DT/AuNCs nanohybrids significantly improved the stability and enhanced the therapeutic efficiency compared to naked DT. Endowing with remarkable stability against biological milieu and site specificity for drug release, our work exhibits a new prospect of fabricating DNA-based nanohybrids for precise tumor theranostics.


Subject(s)
Metal Nanoparticles , Neoplasms , DNA/chemistry , Glutathione , Gold/chemistry , Metal Nanoparticles/chemistry , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , RNA, Messenger/chemistry
15.
Nucleic Acids Res ; 50(18): e106, 2022 10 14.
Article in English | MEDLINE | ID: mdl-35871301

ABSTRACT

With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.


Subject(s)
Endoribonucleases/chemistry , RNA, Messenger/chemistry , Sequence Analysis, RNA/methods , Tandem Mass Spectrometry , Chromatography, Liquid/methods , DNA, Complementary , Humans , Oligonucleotides/analysis , RNA, Messenger/genetics , Ribonuclease T1/metabolism , Tandem Mass Spectrometry/methods
16.
Nucleic Acids Res ; 50(14): 8302-8320, 2022 08 12.
Article in English | MEDLINE | ID: mdl-35808938

ABSTRACT

Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism. We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.


Subject(s)
Peptide Elongation Factor G , Ribosomes , Peptide Elongation Factor G/metabolism , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Transfer/metabolism , Ribosomes/metabolism
17.
ACS Appl Bio Mater ; 5(7): 3476-3486, 2022 07 18.
Article in English | MEDLINE | ID: mdl-35729172

ABSTRACT

Broadening the applicable tools for mRNA delivery provides more flexibility in research and those proven effective and safe can potentially be translated for clinical use. We report here a 27-amino acid peptide sequence mimicking the viral capsid protein, termed pepMAX, capable of co-assembling with mRNA into 100-150 nm nanostructures for efficient transfection of multiple cell lines. The mRNA loading and N/P ratio have been systematically optimized for each cell line. In HeLa, HEK293, and SKNMC, the transfection attained (>80%) is comparable with that of commercially available vectors Lipofectamine MessengerMAXTM (LipoMMAX). Confocal microscopy reveals that pepMAX efficiently delivers mRNA into the cytosol and induces efficient protein production. The pepMAX/mRNA co-assemblies retain their transfection efficiency after storage up to one week at room temperature in lyophilized form.


Subject(s)
Gene Transfer Techniques , Nanostructures , RNA, Messenger , HEK293 Cells , Humans , Nanostructures/chemistry , Oligopeptides/chemistry , RNA, Messenger/chemistry , RNA, Messenger/pharmacology
18.
Aging Cell ; 21(7): e13657, 2022 07.
Article in English | MEDLINE | ID: mdl-35718942

ABSTRACT

With the aging of the global population, accumulating interest is focused on manipulating the fundamental aging-related signaling pathways to delay the physiological aging process and eventually slow or prevent the appearance or severity of multiple aging-related diseases. Recently, emerging evidence has shown that RNA modifications, which were historically considered infrastructural features of cellular RNAs, are dynamically regulated across most of the RNA species in cells and thereby critically involved in major biological processes, including cellular senescence and aging. In this review, we summarize the current knowledge about RNA modifications and provide a catalog of RNA modifications on different RNA species, including mRNAs, miRNAs, lncRNA, tRNAs, and rRNAs. Most importantly, we focus on the regulation and roles of these RNA modifications in aging-related diseases, including neurodegenerative diseases, cardiovascular diseases, cataracts, osteoporosis, and fertility decline. This would be an important step toward a better understanding of fundamental aging mechanisms and thereby facilitating the development of novel diagnostics and therapeutics for aging-related diseases.


Subject(s)
Aging/pathology , MicroRNAs , RNA, Long Noncoding , Cellular Senescence , MicroRNAs/chemistry , RNA, Long Noncoding/chemistry , RNA, Messenger/chemistry
19.
Nat Chem ; 14(8): 905-913, 2022 08.
Article in English | MEDLINE | ID: mdl-35725774

ABSTRACT

The translation of messenger RNA (mRNA) is a fundamental process in gene expression, and control of translation is important to regulate protein synthesis in cells. The primary hallmark of eukaryotic mRNAs is their 5' cap, whose molecular contacts to the eukaryotic translation initiation factor eIF4E govern the initiation of translation. Here we report 5' cap analogues with photo-cleavable groups (FlashCaps) that prohibit binding to eIF4E and resist cleavage by decapping enzymes. These compounds are compatible with the general and efficient production of mRNAs by in vitro transcription. In FlashCap-mRNAs, the single photocaging group abrogates translation in vitro and in mammalian cells without increasing immunogenicity. Irradiation restores the native cap, triggering efficient translation. FlashCaps overcome the problem of remaining sequence or structure changes in mRNA after irradiation that limited previous designs. Together, these results demonstrate that FlashCaps offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control.


Subject(s)
Eukaryotic Initiation Factor-4E , Protein Biosynthesis , Animals , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Mammals/genetics , Mammals/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics
20.
J Mol Biol ; 434(14): 167662, 2022 07 30.
Article in English | MEDLINE | ID: mdl-35640718

ABSTRACT

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3' end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.


Subject(s)
Immediate-Early Proteins , Poly(A)-Binding Proteins , Tumor Suppressor Proteins , Humans , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/genetics , Models, Structural , Molecular Docking Simulation , Mutagenesis , Poly A/chemistry , Poly A/metabolism , Poly(A)-Binding Proteins/chemistry , Poly(A)-Binding Proteins/genetics , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
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