Protocol

mRNA Processing and Metabolism

Volume 257 of the series Methods in Molecular Biology™ pp 171-180

A Poly(A) Tail-Responsive In Vitro System for Cap- or IRES-Driven Translation From HeLa Cells

  • Christian ThomaAffiliated withGene Expression Programme, EMBL
  • , Antje Ostareck-LedererAffiliated withAnadys Pharmaceuticals Europe GmbH
  • , Matthias W. HentzeAffiliated withGene Expression Programme, EMBL

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Abstract

In cells, the poly(A) tail stimulates translation from messenger RNAs bearing a cap structure or viral IRES elements. This 3′ end-mediated stimulation of translation is not reflected in commonly used commercial cell-free translation systems prepared from rabbit reticulocytes or wheat germ. We describe a simple procedure to generate poly(A) tail-responsive translation extracts from HeLa cells. We suggest that this procedure should be adaptable to many animal cell lines.

Key Words

Poly(A) tail cap structure internal ribosome entry site (IRES) translation RNA stability