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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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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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The ER, Golgi apparatus, endosomes, and lysosomes work in tandem to modify, sort, and package proteins and lipids. An integrated membrane trafficking network facilitates the back and forth shuttling of molecules within different organelles in the same cell or across the cell membrane.
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Nuclear Protein Sorting01:34

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Nuclear protein sorting is the selective trafficking of histones, polymerases, gene regulatory proteins into the nucleus and exporting RNAs and ribosomes to the cytosol. It is a tightly controlled process that regulates gene expression within a cell.
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Regulation of Expression Occurs at Multiple Steps02:24

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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.
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Regulation of Nuclear Protein Sorting01:45

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Nuclear protein sorting regulates nucleus composition and gene expression, crucial for determining the fate of a eukaryotic cell. Hence, the entry and exit of molecules across the nuclear envelope is a tightly controlled process. Nuclear protein sorting can be inhibited by one of the following ways: 1) masking cargo signal sequences, 2) modifying the nuclear receptor's affinity for cargo, 3) controlling the nuclear pore size, 4) retaining the cargo during its transit to the cytosol or the...
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Updated: Jun 16, 2025

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
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Inside the cell: Approaches to evaluating mRNA internalization and trafficking.

Claudia Del Toro Runzer1, Christian Plank2, Martijn van Griensven3

  • 1Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht, Limburg 6229 ER, the Netherlands.

Methods (San Diego, Calif.)
|June 13, 2025
PubMed
Summary
This summary is machine-generated.

Accurately measuring messenger RNA (mRNA) cellular uptake is crucial for developing effective mRNA therapeutics. This study offers methods to quantify mRNA internalization in human cells, aiding delivery system optimization.

Keywords:
Cellular uptakeCorrelative light and electron microscopyEndocytic pathwaysTransfer to lysosomesmRNA

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Drug Delivery

Background:

  • Messenger RNA (mRNA) therapeutics and vaccines are rapidly advancing.
  • Efficient cellular uptake is critical for mRNA delivery system efficacy.
  • Understanding mRNA internalization pathways is essential for therapeutic development.

Purpose of the Study:

  • To present methods for quantifying and visualizing mRNA internalization in human primary cells.
  • To evaluate the utility of fluorescent probes for mRNA uptake assessment.
  • To investigate mRNA uptake pathways using endocytic inhibitors.

Main Methods:

  • Transfection of human mesenchymal stromal cells, fibroblasts, and osteoblasts.
  • Utilizing fluorescent probes for qualitative and quantitative mRNA uptake analysis.
  • Employing endocytic inhibitors (chlorpromazine, wortmannin, genistein) to study uptake mechanisms.
  • Applying flow cytometry and correlative light and electron microscopy (CLEM).

Main Results:

  • Demonstrated methods for quantifying mRNA internalization in diverse human cell types.
  • Highlighted the effectiveness of fluorescent probes in assessing mRNA uptake and trafficking.
  • Identified specific endocytic pathways involved in mRNA internalization.

Conclusions:

  • Established robust methodologies for evaluating mRNA cellular uptake.
  • Provided insights into mRNA internalization mechanisms, crucial for optimizing delivery systems.
  • A deeper understanding of mRNA internalization is vital for advancing mRNA-based therapeutics and vaccines.