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Common cell lysis procedures distort ribosome profiling analyses of gene expression
Aoife O'Connell1,2, Alla D Fedorova2, Patrick B F O'Connor2
1School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
Genome Biology
|August 12, 2025
Summary
Centrifugation in ribosome profiling depletes translated mRNAs, particularly those for cytoskeleton proteins. Omitting this step can improve gene expression analysis accuracy.
Area of Science:
- Molecular Biology
- Genomics
- Gene Expression Analysis
Background:
- Ribosome profiling is a key technique for transcriptome-wide gene expression studies.
- It involves sequencing mRNA fragments protected by ribosomes after nuclease digestion.
- Standard protocols often include cell lysis followed by centrifugation and digestion.
Purpose of the Study:
- To investigate the impact of centrifugation on mRNA recovery in ribosome profiling.
- To identify specific mRNA populations affected by the centrifugation step.
- To propose a modification to enhance the accuracy of ribosome profiling.
Main Methods:
- Performing ribosome profiling experiments on HEK293T cells.
- Comparing results from protocols with and without a centrifugation step post-lysis.
- Analyzing the abundance of translated mRNAs using sequencing data.
Main Results:
- Centrifugation was found to deplete 329 translated mRNAs in HEK293T cells.
- A significant proportion of depleted mRNAs encoded cytoskeleton proteins.
- This suggests potential underestimation of certain gene expression levels in standard protocols.
Conclusions:
- The centrifugation step in ribosome profiling can lead to the loss of specific translated mRNAs.
- Cytoskeleton protein-encoding mRNAs are particularly vulnerable to depletion.
- Excluding centrifugation after cell lysis can improve the accuracy of ribosome profiling for these transcripts.
