Related Experiment Video
Updated: Jun 20, 2026
08:53
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
A dual reporter approach to quantify defects in messenger RNA processing.
Ayan Banerjee1, Mimi C Sammarco, Scott Ditch
1Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Analytical Biochemistry
|September 8, 2009
Summary
This study introduces a novel dual reporter system for assessing gene expression defects. The system efficiently detects deficiencies in splicing and nuclear export, crucial for eukaryotic gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Splicing and nuclear export are fundamental processes in eukaryotic gene expression.
- Defects in splicing are linked to various human diseases.
- Understanding cotranscriptional machinery is key to gene expression regulation.
Purpose of the Study:
- To develop a robust, internally controlled dual reporter system for analyzing splicing and nuclear export deficiencies.
- To enable high-throughput screening of factors affecting these processes.
- To investigate the impact of cotranscriptional machinery components on gene expression.
Main Methods:
- Utilizing a bidirectional promoter to coexpress a test reporter (dependent on splicing and export) and an intronless control reporter.
- Implementing a dual reporter system for ratiometric output, normalizing for cell number and transfection efficiency.
- Employing a nonsplicing control construct for accurate assessment.
Main Results:
- The dual reporter system provides a sensitive and reliable output, independent of experimental variations.
- Demonstrated sensitivity to defects in nuclear export by depleting essential cotranscriptional factors (UAP56 and NXF1).
- The system effectively distinguishes between splicing and export-related deficiencies.
Conclusions:
- The developed dual reporter system is a powerful tool for studying gene expression regulation.
- It facilitates high-throughput analysis of splicing and nuclear export functions.
- This system aids in understanding the molecular basis of diseases linked to splicing and export defects.
Related Concept Videos
02:27Nonsense-mediated mRNA Decay
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,...
02:31Nuclear Export of mRNA
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...
02:24Ribosome Profiling
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 helps...
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 helps...
02:28Leaky Scanning
During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA. Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
02:24Regulation of Expression Occurs at Multiple Steps
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...
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...
02:51mRNA Stability and Gene Expression
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