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Related Concept Videos

Regulation of Expression Occurs at Multiple Steps02:24

Regulation of Expression Occurs at Multiple Steps

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Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the binding of proteins to short regulatory sequences on the DNA. This association can either promote or inhibit the transcription of a gene associated with the respective sequence.
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a...
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mRNA Stability and Gene Expression02:51

mRNA Stability and Gene Expression

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The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
Cis-acting Elements involved in mRNA stability
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pre-mRNA Processing02:01

pre-mRNA Processing

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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl...
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Initiation of Translation02:33

Initiation of Translation

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Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
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Regulated mRNA Transport02:22

Regulated mRNA Transport

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In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing...
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Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
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Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells
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Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

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Development and application of an uncapped mRNA platform.

Xiaodi Zheng1, Biao Liu1, Peng Ni2

  • 1College of Bioengineering, National ''111'' Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, China.

Annals of Medicine
|December 9, 2024
PubMed
Summary
This summary is machine-generated.

A novel uncapped mRNA platform offers an economical and efficient alternative for vaccine and protein replacement therapies. This platform, utilizing internal ribosome entry site (IRES) and uridine triphosphate (UTP), successfully induced potent neutralizing antibodies against SARS-CoV-2 variants.

Keywords:
LNPOmicron strain neutralizationUncapped mRNA

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Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA
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Area of Science:

  • Biotechnology
  • Vaccinology
  • Molecular Biology

Background:

  • Development of a novel uncapped messenger RNA (mRNA) platform.
  • Focus on process simplification and economic viability.

Purpose of the Study:

  • Evaluate the transfection efficiency and durability of uncapped mRNA constructs in vitro and in vivo.
  • Assess the activation of humoral and cellular immunity.
  • Compare mRNA constructs synthesized with uridine triphosphate (UTP) versus N1-methylpseudouridine triphosphate (N1mψTP).

Main Methods:

  • Creation of five lipid nanoparticle (LNP)-encapsulated mRNA constructs: eGFP, Fluc, SARS-CoV-2 spike protein (SδT), and two herpes simplex virus type 2 glycoprotein D variants (gDED, gDFR).
  • In vitro and in vivo evaluation of protein expression and immune responses in animal models.
  • Utilized an internal ribosome entry site (IRES) for cap-independent translation.

Main Results:

  • Achieved quantifiable protein expression in vitro and in vivo.
  • Demonstrated comparable humoral and cellular immune responses to capped mRNA constructs.
  • Induced potent neutralizing antibodies against SARS-CoV-2 Delta and Omicron variants with SδT-mRNA-LNP.
  • UTP-based mRNA constructs showed superior antibody induction compared to N1mψTP-based constructs.

Conclusions:

  • The developed uncapped mRNA platform (Rapid, Amplified, Capless and Economical, RACE; BH-RACE) is process-simplified and economical.
  • The platform enables efficient, rapid, and economical mRNA expression in cells via a self-developed packaging instrument and LNP delivery system.
  • Potential broad utility in vaccine development and protein replacement therapies, as evidenced by potent neutralizing antibody induction against SARS-CoV-2 variants.