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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Global analysis of HuR-regulated gene expression in colon cancer systems of reducing complexity
Isabel López de Silanes1, Jinshui Fan, Craig J Galbán
1Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA.
Abstract:
HuR, a protein that binds to target mRNAs and can enhance their stability and translation, is increasingly recognized as a pivotal regulator of gene expression during cell division and tumorigenesis. We sought to identify collections of HuR-regulated mRNAs in colon cancer cells by systematic, cDNA array-based assessment of gene expression in three systems of varying complexity. First, comparison of gene expression profiles among tumors with different HuR abundance revealed highly divergent gene expression patterns, and virtually no changes in previously reported HuR target mRNAs. Assessment of gene expression patterns in a second system of reduced complexity, cultured colon cancer cells expressing different HuR levels, rendered more conserved sets of HuR-regulated mRNAs. However, the definitive identification of direct HuR target mRNAs required a third system of still lower complexity, wherein HuR-RNA complexes immunoprecipitated from colon cancer cells were subject to cDNA array hybridization to elucidate the endogenous HuR-bound mRNAs. Comparison of the transcript sets identified in each system revealed a strikingly limited overlap in HuR-regulated mRNAs. The data derived from this systematic analysis of HuR-regulated genes highlight the value of low-complexity, biochemical characterization of protein-RNA interactions. More importantly, however, the data underscore the broad usefulness of integrated approaches comprising systems of low complexity (protein-nucleic acid) and high complexity (cells, tumors) to comprehensively elucidate the gene regulatory events that underlie biological processes.
Insights
Identifying RNA-binding protein HuR targets in colon cancer requires integrated approaches. Low-complexity methods, like immunoprecipitation, are crucial for pinpointing direct HuR-regulated mRNAs.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- The RNA-binding protein HuR (human antigen R) is a key regulator of mRNA stability and translation.
- HuR plays a significant role in cell division and the development of cancer, particularly colon cancer.
- Understanding HuR's specific mRNA targets is crucial for deciphering its regulatory mechanisms in tumorigenesis.
Purpose of the Study:
- To systematically identify HuR-regulated messenger RNAs (mRNAs) in colon cancer cells.
- To compare gene expression patterns across systems of varying complexity to identify direct HuR targets.
- To evaluate the utility of integrated approaches combining high- and low-complexity systems for studying protein-RNA interactions.
Main Methods:
- Gene expression profiling using cDNA arrays in three systems: tumors with varying HuR levels, colon cancer cells with different HuR expression, and immunoprecipitated HuR-RNA complexes.
- Analysis of gene expression patterns to identify HuR-regulated mRNAs.
- Biochemical characterization of endogenous HuR-bound mRNAs via immunoprecipitation and array hybridization.
Main Results:
- Gene expression profiles in tumors showed highly divergent patterns, with minimal overlap with previously reported HuR targets.
- Cultured colon cancer cells with varying HuR levels yielded more conserved sets of HuR-regulated mRNAs.
- Direct HuR target mRNAs were definitively identified using low-complexity immunoprecipitation of HuR-RNA complexes, revealing limited overlap between methods.
Conclusions:
- A combination of high- and low-complexity systems is essential for comprehensively elucidating gene regulatory events.
- Low-complexity biochemical characterization of protein-RNA interactions, such as HuR-RNA immunoprecipitation, is highly valuable.
- Integrated approaches are critical for accurately identifying direct mRNA targets of regulatory proteins like HuR in complex biological processes such as cancer.
