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Translation factors promote the formation of two states of the closed-loop mRNP

Abstract

Efficient translation initiation and optimal stability of most eukaryotic messenger RNAs depends on the formation of a closed-loop structure and the resulting synergistic interplay between the 5′ m7G cap and the 3′ poly(A) tail1,2. Evidence of eIF4G and Pab1 interaction supports the notion of a closed-loop mRNP3, but the mechanistic events that lead to its formation and maintenance are still unknown. Here we use toeprinting and polysome profiling assays to delineate ribosome positioning at initiator AUG codons and ribosome–mRNA association, respectively, and find that two distinct stable (resistant to cap analogue) closed-loop structures are formed during initiation in yeast cell-free extracts. The integrity of both forms requires the mRNA cap and poly(A) tail, as well as eIF4E, eIF4G, Pab1 and eIF3, and is dependent on the length of both the mRNA and the poly(A) tail. Formation of the first structure requires the 48S ribosomal complex, whereas the second requires an 80S ribosome and the termination factors eRF3/Sup35 and eRF1/Sup45. The involvement of the termination factors is independent of a termination event.

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Figure 1: Toeprint analyses of initiation on long and short mRNAs in the presence of cycloheximide in wild-type extracts.
Figure 2: Cap analogue resistance of the miniUAA1 mRNA is dependent on cap and poly(A) in wild-type extracts and suggests formation of a stable closed-loop structure.
Figure 3: Formation of a stable closed-loop structure on a capped and polyadenylated mRNA in the presence of an 80S complex requires Pab1 interactions with eIF4G, mRNA, and Sup35.
Figure 4: Stabilization of the closed-loop structure on a capped and polyadenylated mRNA in the presence of a 48S complex requires interactions of Pab1 with eIF4G and mRNA.

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Acknowledgements

We thank S. Kervestin for providing us with recombinant Pab1; J. McCarthy for eIF4E antibodies; A. Hinnebusch for the cdc33 strain; D. Bedwell for the plasmid-borne sup35-R419G allele; and members of the Jacobson laboratory for comments and discussions. This work was supported by a grant to A.J. from the National Institutes of Health.

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Correspondence to Allan Jacobson.

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The file contains Supplementary Data, Supplementary Methods with additional references, Supplementary Table 1 and Supplementary Figures 1-4 with Legends. (PDF 2678 kb)

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Amrani, N., Ghosh, S., Mangus, D. et al. Translation factors promote the formation of two states of the closed-loop mRNP. Nature 453, 1276–1280 (2008). https://doi.org/10.1038/nature06974

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