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Nature Apr 2023Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for...
Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export. However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood. Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export.
Topics: Humans; Active Transport, Cell Nucleus; Cell Nucleus; Cryoelectron Microscopy; RNA, Messenger; Transcription, Genetic; Exons
PubMed: 37020021
DOI: 10.1038/s41586-023-05904-0 -
Biophysical Journal Jul 2021Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is...
Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphology. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid associated.
Topics: Cryoelectron Microscopy; Lipids; Nanoparticles; RNA, Messenger
PubMed: 33773963
DOI: 10.1016/j.bpj.2021.03.012 -
BioTechniques Jul 2005Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly... (Review)
Review
Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.
Topics: Algorithms; Chemistry Techniques, Analytical; Gene Expression Profiling; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Fluorescence
PubMed: 16060372
DOI: 10.2144/05391RV01 -
Advanced Materials (Deerfield Beach,... Feb 2019Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro...
Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.
Topics: Administration, Inhalation; Animals; Drug Compounding; Drug Liberation; Epithelial Cells; Female; Gene Transfer Techniques; Genetic Therapy; Hydrogen-Ion Concentration; Luciferases; Lung; Mice; Mice, Inbred C57BL; Models, Animal; Nanoparticles; Polymers; RNA, Messenger; Tissue Distribution; Transfection
PubMed: 30609147
DOI: 10.1002/adma.201805116 -
Nature Biotechnology Jul 2019The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome...
The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.
Topics: 5' Untranslated Regions; Base Sequence; Gene Expression Regulation; Humans; Models, Genetic; Protein Biosynthesis; Pseudouridine; RNA, Messenger; Reproducibility of Results; Ribosomes
PubMed: 31267113
DOI: 10.1038/s41587-019-0164-5 -
Molecular Therapy : the Journal of the... Jun 2018The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option....
The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.
Topics: Animals; Lipids; Nanoparticles; Primates; RNA, Messenger; Rats
PubMed: 29653760
DOI: 10.1016/j.ymthe.2018.03.010 -
Wiley Interdisciplinary Reviews. RNA Jan 2017RNA modifications are indispensable for the translation machinery to provide accurate and efficient protein synthesis. Whereas the importance of transfer RNA (tRNA) and... (Review)
Review
RNA modifications are indispensable for the translation machinery to provide accurate and efficient protein synthesis. Whereas the importance of transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications has been well described and is unquestioned for decades, the significance of internal messenger RNA (mRNA) modifications has only recently been revealed. Novel experimental methods have enabled the identification of thousands of modified sites within the untranslated and translated regions of mRNAs. Thus far, N -methyladenosine (m A), pseudouridine (Ψ), 5-methylcytosine (m C) and N -methyladenosine (m A) were identified in eukaryal, and to some extent in prokaryal mRNAs. Several of the functions of these mRNA modifications have previously been reported, but many aspects remain elusive. Modifications can be important factors for the direct regulation of protein synthesis. The potential diversification of genomic information and regulation of RNA expression through editing and modifying mRNAs is versatile and many questions need to be addressed to completely elucidate the role of mRNA modifications. Herein, we summarize and highlight some recent findings on various co- and post-transcriptional modifications, describing the impact of these processes on gene expression, with emphasis on protein synthesis. WIREs RNA 2017, 8:e1375. doi: 10.1002/wrna.1375 For further resources related to this article, please visit the WIREs website.
Topics: 5-Methylcytosine; Animals; Humans; Methylation; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcriptome
PubMed: 27345446
DOI: 10.1002/wrna.1375 -
Communications Biology Aug 2021Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP...
Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.
Topics: Administration, Intravenous; Animals; Drug Compounding; Gene Expression; Humans; Hydrogen-Ion Concentration; Injections, Intramuscular; Ions; Lipids; Mice; Molecular Structure; Nanoparticles; RNA, Messenger; Spectrum Analysis; Tissue Distribution; Transfection
PubMed: 34381159
DOI: 10.1038/s42003-021-02441-2 -
BMC Biology Dec 2015Zebrafish are able to regenerate various organs and tissues after damage or amputation. To understand better the genetic controls of this process, the authors of this...
Zebrafish are able to regenerate various organs and tissues after damage or amputation. To understand better the genetic controls of this process, the authors of this study investigated the expression of two genes previously implicated in fin regeneration using semi-quantitative RT-PCR, at three time points after fin amputation (T1, T2, and T3 in Fig. 1, corresponding to the initiation, middle, and end of fin regeneration, respectively). Briefly, the RT-PCR procedure involved isolating messenger RNA (mRNA) from a matched amount of zebrafish cells from the site of fin regeneration at the three time points, and using primers specific to each gene to selectively detect mRNA as an indicator of gene expression levels. The authors used total genomic DNA isolated from zebrafish cells as a positive control, and no RNA or DNA template as a negative control. They found that Gene 1 was only expressed early on in the process, while Gene 2 expression gradually increased during fin regeneration, reaching a peak of expression toward the end of the process. This provides some detailed information that could be useful in elucidating the function of these genes in fin regeneration.
Topics: Amputation, Surgical; Animal Fins; Animals; Gene Expression Regulation; RNA, Messenger; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish
PubMed: 26694721
DOI: 10.1186/s12915-015-0217-2 -
Cell Mar 2015mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate...
mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.
Topics: Codon; Protein Biosynthesis; RNA Stability; RNA, Fungal; RNA, Messenger; Saccharomyces cerevisiae
PubMed: 25768907
DOI: 10.1016/j.cell.2015.02.029