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Updated: May 12, 2026
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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing
Erik Holmqvist1, Johan Reimegård, E Gerhart H Wagner
1Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, SciLifeLab Uppsala, Box 596, S-75124 Uppsala, Sweden.
Nucleic Acids Research
|April 24, 2013
Summary
This study introduces a novel method combining mutagenesis, cell sorting, and sequencing for rapid functional screening of RNA regulatory elements. The approach efficiently identifies key nucleotide positions impacting gene expression and regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Post-transcriptional gene regulation is crucial for cellular function.
- Small RNAs (sRNAs) play a significant role in modulating gene expression.
- Understanding RNA structure-function relationships is essential for deciphering gene regulation.
Purpose of the Study:
- To develop and validate a high-throughput method for functional screening of RNA regulatory sequences.
- To investigate the impact of mutations on the post-transcriptional regulation of bacterial csgD messenger RNA (mRNA).
- To identify functionally important nucleotide positions within an mRNA sequence that influence gene expression and sRNA-mediated control.
Main Methods:
- Random mutagenesis of a 109 bp sequence within the csgD 5'-untranslated region (5'-UTR).
- Monitoring gene expression using a translational green fluorescent protein (GFP) fusion.
- Phenotypic cell sorting based on distinct expression levels using fluorescence-activated cell sorting (FACS).
- High-throughput sequencing of mutant plasmids from sorted cell populations.
Main Results:
- Identification of functionally important nucleotide positions affecting intrinsic translational activity.
- Pinpointing mutations that alter extrinsic, sRNA-based gene regulation.
- Distinguishing between mutations that alter gene expression and those that are silent.
- Validation of previously published data on csgD mRNA regulation.
Conclusions:
- The developed method enables efficient functional screening of numerous mutations in a single experiment.
- This approach is valuable for studying RNA structure-function relationships, including sRNA-mediated control and riboswitch elements.
- The protocol can be adapted for mapping functional DNA elements and protein regions.
