Related Experiment Video
Updated: Jan 21, 2026
11:32
Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
Published on: December 4, 2010
15.8K
Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System
Maria Vera1, Evelina Tutucci2, Robert H Singer3,4
1Department of Biochemistry, McGill University, Montreal, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|August 14, 2019
Summary
This study presents an improved protocol for visualizing single messenger RNAs (mRNAs) in mammalian cells using the latest MS2-MCP system versions, MS2V6 and MS2V7. MS2V7 offers enhanced reporter properties for live-cell imaging, advancing gene expression studies.
Area of Science:
- Molecular Biology
- Cell Biology
- Biophysics
Background:
- Visualizing single messenger RNAs (mRNAs) in real-time is crucial for understanding gene expression regulation.
- The MS2-MS2-Capsid Protein (MCP) system has been instrumental in tagging and imaging mRNA dynamics.
- Previous MS2-MCP systems faced limitations, including mRNA degradation issues in certain organisms.
Purpose of the Study:
- To provide an updated protocol for utilizing the advanced MS2V6 and MS2V7 systems for mRNA visualization in mammalian cells.
- To address and overcome previously identified mRNA degradation caveats.
- To evaluate and recommend the optimal MS2-MCP system version for live-cell imaging in mammalian systems.
Main Methods:
- Development and application of the MS2V6 and MS2V7 mRNA tagging systems.
- Utilizing widefield fluorescence microscopy for live-cell imaging.
- Culturing mammalian cells for experimental procedures.
Main Results:
- The MS2V6 and MS2V7 systems were engineered to improve upon earlier MS2-MCP iterations.
- MS2V7 demonstrated superior reporter properties specifically for live-cell imaging in mammalian cells.
- The protocol facilitates enhanced qualitative and quantitative analysis of mRNA localization and dynamics.
Conclusions:
- The MS2V7 system is recommended for live-cell mRNA imaging in mammalian cells due to its improved performance.
- This protocol enables robust visualization of single mRNAs, aiding in the study of gene expression.
- The advancements in the MS2-MCP system continue to enhance our ability to investigate cellular processes at the molecular level.
Related Concept Videos
02:01pre-mRNA Processing
57.2K
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...
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...
57.2K
02:31Nuclear Export of mRNA
8.7K
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...
8.7K
02:27Nonsense-mediated mRNA Decay
11.7K
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,...
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
11.7K
02:22Regulated mRNA Transport
6.9K
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...
6.9K
02:51mRNA Stability and Gene Expression
6.5K
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
Cis-acting Elements involved in mRNA stability
6.5K
02:41Chromatin Structure Regulates pre-mRNA Processing
8.1K
In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
The chromatin structure, especially...
The chromatin structure, especially...
8.1K