A new plant protein interacts with eIF3 and 60S to enhance virus‐activated translation re‐initiation

The plant viral re‐initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re‐initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor—re‐initiation supporting protein (RISP)—that enhances TAV function in re‐initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C‐terminus of ribosomal protein L24. RISP pre‐bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co‐localize in epidermal cells of infected plants, and eIF3–TAV–RISP–L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3‐bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.     

Analysis of the interacting partners eIF4F and 3′‐CITE required for Melon necrotic spot virus cap‐independent translation

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3′‐cap‐independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV‐Mα5 3′‐CITE and its translation initiation factor partner. We first mapped the minimal 3′‐CITE (Ma5TE) to a 45‐nucleotide sequence, which consists of a stem‐loop structure with two internal loops, similar to other I‐shaped 3′‐CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G_980–1159 (eIF4F_p20), but not with each subunit alone or with eIF4E + eIF4G_1003–1092, suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G_980–1159, or through a double interaction with eIF4E and eIF4G_980–1159. Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G_1003–1092 and cap‐independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE‐driven cap‐independent translation, showing that conserved amino acids in a positively charged RNA‐binding motif around amino acid position 228, implicated in eIF4E–eIF4G binding or belonging to the cap‐recognition pocket, are essential for cap‐independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.     

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA^fMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.