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J Vis Exp. 2014 May 17;(87). doi: 10.3791/51455.

Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.

Author information

1
Lady Davis Institute and Department of Oncology, McGill University.
2
Department of Oncology-Pathology, Karolinska Institutet.
3
Goodman Cancer Centre and Department of Biochemistry, McGill University.
4
Department of Oncology-Pathology, Karolinska Institutet; ola.larsson@ki.se.
5
Lady Davis Institute and Department of Oncology, McGill University; ivan.topisirovic@mcgill.ca.

Abstract

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.

PMID:
24893926
PMCID:
PMC4189431
DOI:
10.3791/51455
[Indexed for MEDLINE]
Free PMC Article
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