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Ribo Mega-SEC Provides a Rapid, Efficient and Reproducible Analysis of Mammalian Polysomes and Ribosomal Subunits

ABSTRACT: We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC, which was achieved within 15 min from sample injection to fraction collection. Ribo Mega-SEC reproducibly shows translating ribosomes exist predominantly in polysome complexes in extracts isolated from human cell lines and mouse liver tissue, which alter in response to starvation. Ribo Mega-SEC provides a rapid, efficient, convenient and highly reproducible method for studying functional translation complexes and is easily combined with high-through put analysis such as proteomics and RNA-Seq, or with structural analysis using electron microscopy. We propose that Ribo Mega-SEC analysis is an accessible alternative to traditional polysome profiling using sucrose density gradients.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Mark Larance

LAB HEAD: Mark Larance

PROVIDER: PXD008913 | Pride | 2018-08-10

REPOSITORIES: Pride

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Project description:Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (M?), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as wound healing, obesity, and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. To address this, we developed a strategy for simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression in response to DEN and found that while there was only a marginal increase in hepatocyte number, M? and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, M? and SEC identified characteristic gene “fingerprints” that define each cell type and their distinct physiological or oncogenic stress signatures. Integration of these cell-specific gene fingerprints with available hepatocellular carcinoma (HCC) patient microarray data demonstrates that the hepatocyte-specific response strongly correlates with the human HCC gene expression profile. Liver-specific M? and SEC gene signatures demonstrate significant alterations in inflammatory and angiogenic gene regulatory pathways, which may impact the hepatocyte response to oncogenic stress. Further validation confirms alterations in components of two key pathways, AP-1 and p53, that have been previously associated with HCC onset and progression. Our data reveal unique gene expression patterns that serve as molecular “fingerprints” for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the development of hepatic cancer. Total RNA isolated from each specific cell type (Hepatocyte (PH), Sinusoidal Endothelial Cell (SEC), and Macrophage (MP)) pooled from 3-4 DEN-treated mice and compared to their untreated counterparts.

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Ribo Mega-SEC Provides a Rapid, Efficient and Reproducible Analysis of Mammalian Polysomes and Ribosomal Subunits

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