Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

Abstract

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

“…A small portion of the protein also cosediments with the 80S and polysome fractions. These results are consistent with the observation that tagged Sbp1 was pulled down with 80S and polysomes (Zhang et al 2014;Wang et al 2016).…”

“…Thus, the distinctive RRM domain architecture in Pab1 and Sbp1 will most likely lead to different conformations when these two proteins interact with the same RNA and, as a result, remodel the RNA structure differently. A related question is whether Sbp1 prefers binding to specific transcripts containing poly(A) or perhaps even the same transcript in different stages of mRNA biogenesis, considering that Sbp1 also binds to mRNA sequences other than poly(A) (Mitchell et al 2013;Wang et al 2016). Clearly, further studies involving genomic and structural investigations are required to answer these questions.…”

Translational regulation in response to stress in Saccharomyces cerevisiae

“…Indeed, some highly translated mRNAs are relatively depleted for them, suggesting the existence of alternative mechanisms to promote their translation (Costello et al, ). On the other hand, these data could indicate that there is a step in the standard initiation pathway where eIF4E and eIF4G are less stably bound to mRNAs, such as during 60S joining (Amrani, Ghosh, Mangus, & Jacobson, ; Wang et al, ). Recently it was shown that many mRNAs show similar reciprocal changes in translational efficiency and binding to both eIF4E and eIF4G during glucose starvation, amino acid starvation and oxidative stress (Costello et al, ).…”

Multiple discrete soluble aggregates influence polyglutamine toxicity in a Huntington’s disease model system

“…We have consequently used the technique of analytical ultracentrifugation with fluorescent detection (AU-FDS) 29 30 to identify Htt soluble aggregates using the yeast HD model system. AU-FDS allows the identification of the sizes and abundances of soluble macromolecular complexes ranging in sizes up to at least 1000S (about 160 MDa) 31 32 33 34 . It allows the extremely rapid and precise (at least an order of magnitude better than sucrose gradient analysis) determination of the size of protein complexes.…”

“…Because we have previously demonstrated that AU-FDS allows specific detection, discrimination, size determination, and relative quantitation of all macromolecular complexes containing specific proteins 31 32 33 34 , we have determined which complexes Htt-polyQ proteins can form. The particular advantage of AU-FDS analysis is that it allows one to identify the complexes of a fluorescently tagged molecule (GFP in this case) in an impure mixture or cellular extract.…”