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Regulated mRNA Transport

Regulated mRNA Transport

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 specific...

Regulated mRNA Transport

Regulated mRNA Transport

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 specific...

Ribosome Profiling

Ribosome 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...

pre-mRNA Processing

pre-mRNA Processing

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 guanosine). This 5’ cap helps the...

Nuclear Export of mRNA

Nuclear 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...

mRNA Stability and Gene Expression

mRNA 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

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Related Experiment Video

Updated: May 15, 2026

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Published on: September 15, 2020

Streamlined protocol for mRNA display.

Pamela A Barendt¹, Daphne T W Ng, Casey N McQuade

¹Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd Street, Philadelphia, Pennsylvania 19104-6321, United States.

ACS Combinatorial Science

|January 12, 2013

Summary

This study streamlines messenger RNA (mRNA) display for protein evolution. The new protocol significantly reduces experiment time and complexity, making directed evolution more accessible.

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Last Updated: May 15, 2026

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

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Published on: September 15, 2020

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

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Published on: September 16, 2019

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Published on: December 9, 2022

Area of Science:

Biochemistry
Molecular Biology
Protein Engineering

Background:

Messenger RNA (mRNA) display is a key technique for in vitro directed evolution of polypeptides.
Current mRNA display protocols are lengthy and technically demanding, limiting their broad application.

Purpose of the Study:

To develop a streamlined and accelerated protocol for mRNA display.
To simplify the technical requirements of mRNA display experiments.

Main Methods:

Replaced lengthy mRNA purification with precipitation and ultrafiltration.
Utilized a reconstituted translation system to eliminate intermediate purification steps.
Performed reverse transcription post-selection to protect polypeptides and further simplify the process.

Main Results:

Successfully recovered the Her2-binding designed ankyrin repeat protein (DARPin) using binary libraries.
Achieved enrichment factors of up to 2 orders of magnitude per selection round.
Reduced the time for one round of selection from approximately 4-7 days to 2 days.

Conclusions:

The developed protocol significantly simplifies and accelerates mRNA display.
This optimized method enhances the accessibility and efficiency of in vitro directed evolution for polypeptide discovery.