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' m(7)G cap and the 3' poly(A) tail. Evidence of eIF4G and Pab1 interaction supports the notion of a closed-loop mRNP, 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.

Fig. 1

Toeprint analyses of initiation on long and short mRNAs in the presence of CHX in wild-type extracts. (a) General schematic of the miniUAA1, UAA, and AAA mRNAs. Sizes (in nt) listed under each construct respectively refer to the length of the 5′ UTR, the coding region, the 3′ UTR, and the poly(A) tail. (b) Addition of cap analog to AAA and UAA mRNAs inhibits accumulation of the AUG toeprint bands. (c) miniUAA1 mRNA AUG toeprints are resistant to 2.7mM cap analog and become sensitive only at higher concentrations. (d) Sensitivity to 2.7 mM cap analog is mRNA size dependent. The values on the graph are the average of two independent experiments. Positions of the toeprints are indicated with arrows. The left portions of panels b and c show dideoxynucleotide sequencing reactions for the AAA or miniUAA1 templates (with 5′ to 3′ sequence reading from the top to the bottom).

Fig. 2

Cap analog resistance of the miniUAA1 mRNA is cap and poly(A) dependent in wild-type extracts and suggests formation of a stable closed loop structure. (a) AUG toeprint analyses of uncapped mRNA. (b) Poly(A)-deficient mRNAs are highly sensitive to cap analog. (c) Resistance to cap analog is poly(A) size dependent.

Fig. 3

Formation of a stable closed-loop structure on a capped and polyadenylated mRNA in the presence of an 80S complex requires Pab1p interactions with eIF4G, mRNA, and Sup35p. (a) Toeprinting analyses of miniUAA1 mRNA in Pab1p-defective, wild-type, or pbp1Δ extracts. (b) Sensitivity to cap analog in an eIF4G1 mutant incapable of Pab1p interaction. (c) sup35-R419G and sup45-2 mutants show sensitivity to cap analog and additional toeprint bands upstream of the initiator AUG. (d) Sensitivity to cap analog is independent of the termination event. (e, f) Cap analog resistance or sensitivity of miniUAA1 mRNA appears at the onset of translation. (g) Sucrose gradient fractionation of miniUAA1 mRNA translated in wild-type and mutant extracts in the absence and presence of cap analog, and in the presence of CHX. In panel g, the values above the horizontal line depict fraction numbers and those below the line denote the respective percentages of mRNA associated with polysomal or non-polysomal fractions.

Fig. 4

Stabilization of the closed-loop structure on a capped and polyadenylated mRNA in the presence of a 48S complex requires Pab1p interactions with eIF4G and mRNA. (a) Extracts from Pab1p- and eIF4G1-deficient strains are cap analog sensitive in the presence of GMP-PNP. (b) Addition of recombinant Pab1p restores resistance to cap analog in pab1ΔpbpΔ extracts in the presence of GMP-PNP. (c) Extracts from the sup35-R419G and sup45-2 termination mutants are cap analog resistant in the presence of GTP analog.