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Updated: May 20, 2025
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Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Published on: October 28, 2014
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Protocol to perform polysome profiling in primary differentiating murine adipocytes
Mirian Krystel De Siqueira1, Zaynab Nouhi2, Yutian Zhao1
1Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
STAR Protocols
|May 2, 2025
Summary
We developed a polysome profiling protocol to study how protein synthesis is regulated in differentiating mouse fat cells. This method helps understand gene expression changes in metabolic diseases like obesity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Adipogenesis involves complex gene regulation.
- Understanding translational control is crucial for metabolic research.
- Existing methods may not fully capture dynamic changes in protein synthesis during adipocyte differentiation.
Purpose of the Study:
- To present a detailed protocol for polysome profiling in differentiating mouse adipocytes.
- To enable the analysis of actively translating messenger RNAs (mRNAs).
- To investigate translational gene regulation in the context of adipogenesis.
Main Methods:
- Isolation of polysome-bound mRNA from mouse stromal vascular fraction.
- Preparation of cell lysates.
- Ultracentrifugation through sucrose gradients.
- RNA sequencing and motif enrichment analysis.
Main Results:
- The protocol successfully isolates polysome-bound mRNA.
- Enables identification of actively translated genes during adipocyte differentiation.
- Provides insights into translational control mechanisms.
Conclusions:
- This protocol offers a robust method for studying translational regulation in adipocytes.
- Facilitates research into metabolic diseases, obesity, and related cellular phenotypes.
- Links translational control to cellular function and metabolic status.
