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Published online before print March 26, 2008, 10.1261/rna.971208
RNA (2008), 14:960-969. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.
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Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

Lisa Lindqvist1, Hiroaki Imataka2, and Jerry Pelletier1,3

1 Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Quebec H3G 1Y6, Canada
2 RIKEN Genomic Sciences Center, Tsurumi-ku, Yokohama 230-0045, Japan
3 McGill Cancer Centre, Faculty of Medicine, McGill University, Montreal, Quebec H3G 1Y6, Canada

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.

Keywords: protein–mRNA interactions; eIF4F; eIF4H; eIF3; 4-thiouridine

Received December 21, 2007 ; accepted February 15, 2008.


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