Published online 28 March 2012 Nucleic Acids Research, 2012, Vol. 40, No. 13 6199–6207 doi:10.1093/nar/gks278 The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase Yingjie Sun 1 , Evrim Atas1, Lisa Lindqvist2, Nahum Sonenberg2, Jerry Pelletier2 and Amit Meller1,* 1Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA and 2Department of Biochemistry and The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC H3G 1Y6, Canada Received January 16, 2012; Revised and Accepted March 13, 2012 ABSTRACT Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal transla- tion initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 50-UTR. Single molecule fluorescence reson- ance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can signifi- cantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-cS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/ eIF4H can bind directly to loop structures to desta- bilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applic- able to a myriad of DEAD-box helicases employed in RNA metabolism. INTRODUCTION mRNA translation in eukaryotic cells begins with the recruitment of a ribosome to the 50-untranslated region (UTR) of the transcript. This process is regulated by a set of translation factors, and is known as the initiation step (1). In what is thought to be the rate-limiting step of translation, 43S ribosome pre-initiation complexes (40S subunits and associated factors) are recruited to the 50 m7GpppN cap structure—a structural feature present on all nuclear-transcribed eukaryotic mRNAs. This process is mediated by the heterotrimeric cap-binding initiation complex, eukaryotic initiation factor eIF4F, which is composed of a cap-binding factor (eIF4E), an RNA helicase (eIF4A) and a scaffolding protein (eIF4G) (2). Ribosome recruitment occurs as a consequence of inter- actions between eIF4G and the multi-subunit, ribosome-bound eIF3 (3,4). Translation initiation of some eukaryotic mRNAs can occur independently of the 50-end by recruitment of 40S ribosomal subunits to internal ribosome entry sites (IRESs) (5,6). Secondary structure within the 50-UTR poses a barrier to cap-dependent translation, requiring helicase activity to facilitate ribosome recruitment (7). eIF4A-mediated unwinding of RNA is required for ribosome recruitment to mRNA templates with even a modest amount of sec- ondary structure (8) and is thought to allow eIF4F to gain access to the cap structure and/or to restructure cap-proximal RNA sequences (9). The importance of this step in translational regulation is highlighted by the fact that deregulated eIF4F activity, either as a conse- quence of altered subunit levels or perturbed eIF4E regu- lation, is linked to alterations in the mRNA translation proﬁle, with profound effects on the resulting cell proteome (10). Deregulated eIF4F activity has been linked to tumor initiation and maintenance (11–14); there- fore, understanding eIF4A’s helicase properties and mode of action is important for developing strategies to modulate translation initiation and provide novel thera- peutic strategies to target unregulated translation in cancer cells (15,16). *To whom correspondence should be addressed. Tel: +1 617 358 4338; Fax: +1 617 358 2835; Email: ameller@bu.edu The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.