Protocol for monitoring mRNA translation and degradation in human cell-free lysates
These videos have been matched automatically. Contact us if they are not relevant.
These videos have been matched automatically. Contact us if they are not relevant.
View abstract on PubMed
Summary
This summary is machine-generated.This study details a human cell-free translation protocol to monitor messenger RNA (mRNA) translation and degradation. This method aids in analyzing protein synthesis and mRNA stability for various RNA elements.
Area Of Science
- Molecular Biology
- Biochemistry
- Genetics
Background
- Cell-free translation systems offer a controlled environment for studying protein synthesis.
- Monitoring mRNA translation and degradation is crucial for understanding gene expression regulation.
- Existing methods may have limitations in analyzing diverse RNA elements or specific regulatory sequences.
Purpose Of The Study
- To present a detailed protocol for a human cell-free translation system.
- To enable the monitoring of mRNA translation efficiency and degradation rates.
- To facilitate the comparative analysis of different 5' untranslated regions (5' UTRs) and viral RNA elements.
Main Methods
- Preparation of translation-competent human cell lysates using dual centrifugation.
- Generation and capping of reporter mRNAs suitable for luciferase assays.
- Execution of cell-free translation reactions and assessment of mRNA stability via northern blotting.
Main Results
- The protocol provides a robust method for quantifying translation efficiency.
- It allows for the accurate measurement of mRNA decay rates in a cell-free environment.
- The system supports the investigation of sequence-specific effects on translation and stability.
Conclusions
- This human cell-free translation protocol is a valuable tool for studying mRNA fate.
- It enables detailed analysis of regulatory elements influencing protein synthesis and mRNA stability.
- The protocol can be applied to diverse research questions in molecular biology and virology.
These videos have been matched automatically. Contact us if they are not relevant.
These videos have been matched automatically. Contact us if they are not relevant.
Related Concept Videos
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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...
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...
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
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,...