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Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
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An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes
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Tactics targeting circular mRNA biosynthesis.

Xinjie Chen1,2, Chen Wang1,2, Yuan Lu1,2

  • 1Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, Beijing, China.

Biotechnology and Bioengineering
|May 1, 2023
PubMed
Summary
This summary is machine-generated.

Circular messenger RNA (mRNA) offers enhanced stability over traditional mRNA for vaccines. This study optimized circRNA synthesis using T4 Rnl 2 ligation and identified an IRES sequence for efficient translation in mammalian cells.

Keywords:
circular mRNAenzymatic ligationinternal ribosome entry sitemRNA synthesispermuted intron-exon

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Messenger RNA (mRNA) technology is advancing rapidly in medicine and vaccines.
  • Circular mRNA (circRNA) presents a more stable alternative to linear mRNA due to its secondary structure.
  • Current circRNA synthesis methods, relying on linear precursor ligation, suffer from low efficiency.

Purpose of the Study:

  • To explore and optimize circRNA biosynthesis strategies using ribozyme catalysis and enzymatic ligation.
  • To identify efficient internal ribosome entry site (IRES) sequences for enhanced circRNA translation in mammalian cells.
  • To provide guidance for the industrial production of circRNAs and the development of mRNA vaccines.

Main Methods:

  • Screening of group I intron self-splicing sequences, identifying the thymidylate synthase (Td) gene from phage T4 for ribozyme-mediated ligation.
  • Optimization of enzymatic ligation using T4 Rnl 2 with a 20-bp homologous arm.
  • Screening of internal ribosome entry site (IRES) sequences for high ribosome binding and translation efficiency in mammalian cells.

Main Results:

  • Phage T4 thymidylate synthase (Td) ribozyme demonstrated high ligation efficiency.
  • T4 Rnl 2 enzymatic ligation, with a 20-bp homologous arm, achieved the highest ligation efficiency.
  • circRNA produced via T4 Rnl 2 ligation showed an 86% higher expression level compared to Td ribozyme ligation.
  • Mud crab dicistrovirus IRES was identified as a highly effective IRES for efficient and stable circRNA translation in mammalian cells.

Conclusions:

  • Optimized circRNA synthesis strategies using T4 Rnl 2 ligation and Td ribozyme provide efficient methods for circRNA production.
  • The identified mud crab dicistrovirus IRES enables robust circRNA translation in mammalian systems.
  • These advancements are crucial for the industrial-scale production of circRNAs and the future development of mRNA vaccines and therapeutics.