期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 87, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/51455
关键词
Biochemistry; Issue 87; Cells; Eukaryota; Nutritional and Metabolic Diseases; Neoplasms; Metabolic Phenomena; Cell Physiological Phenomena; mRNA translation; ribosomes; protein synthesis; genome-wide analysis; translatome; mTOR; eIF4E; 4E-BP1
资金
- Canadian Institutes of Health Research (CIHR) [MOP-115195]
- FRQ-S
- CIHR Young Investigator Award
- Swedish Research Council
- Swedish Cancer Society
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed anota algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
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