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Published on: February 21, 2014
Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Accompanied with Radioresistance in
Abstract:
Epitranscriptomic reader, writer, and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play crucial roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography-parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitranscriptomic RWE proteins. We also applied the LC-PRM method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.e., MDA-MB-231 and MCF-7 cells and their corresponding radioresistant C5 and C6 clones), with the goal of assessing the roles of these proteins in radioresistance. We found that eight epitranscriptomic RWE proteins were commonly altered by over 1.5-fold in the two pairs of breast cancer cells. Among them, TRMT1 (an m2,2G writer) may play a role in promoting breast cancer radioresistance due to its clinical relevance and its correlation with DNA repair gene sets. To our knowledge, this is the first report of a targeted proteomic method for comprehensive quantifications of epitranscriptomic RWE proteins. We envision that the LC-PRM method is applicable for studying the roles of these proteins in the metastatic transformation of cancer and therapeutic resistance of other types of cancer in the future.
Insights
This study introduces a new proteomic method to measure 152 epitranscriptomic proteins. The method identified key proteins, including TRMT1, that may influence breast cancer radioresistance.
Area of Science:
- Proteomics
- Molecular Biology
- Cancer Research
Background:
- Epitranscriptomic reader, writer, and eraser (RWE) proteins regulate RNA modifications, impacting RNA processing, splicing, and stability.
- Understanding the role of RWE proteins in cancer, particularly in therapeutic resistance, is crucial for developing new treatments.
Purpose of the Study:
- To establish a high-throughput liquid chromatography-parallel-reaction monitoring (LC-PRM) method for profiling 152 epitranscriptomic RWE proteins.
- To quantify RWE protein alterations in radioresistant breast cancer cells using LC-PRM combined with stable isotope labeling by amino acids in cell culture (SILAC).
- To identify RWE proteins involved in breast cancer radioresistance.
Main Methods:
- Development and application of a targeted proteomic LC-PRM assay for comprehensive quantification of 152 epitranscriptomic RWE proteins.
- Utilized SILAC for quantitative proteomic analysis in matched parental and radioresistant breast cancer cell lines (MDA-MB-231 and MCF-7).
- Statistical analysis to identify significantly altered RWE proteins (over 1.5-fold change) between parental and radioresistant cells.
Main Results:
- Successfully profiled 152 epitranscriptomic RWE proteins using the developed LC-PRM method.
- Identified eight RWE proteins commonly altered in two pairs of breast cancer cells exhibiting radioresistance.
- TRMT1, an m²2G writer, was found to be significantly altered and correlated with DNA repair gene sets, suggesting a role in promoting breast cancer radioresistance.
Conclusions:
- The developed LC-PRM method provides a powerful tool for high-throughput quantification of epitranscriptomic RWE proteins.
- TRMT1 is a potential key player in breast cancer radioresistance, warranting further investigation.
- This proteomic approach can be extended to study RWE proteins in other cancer types and their roles in metastasis and therapeutic resistance.
