Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation

Abstract

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2β and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2β, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.

Principles, mechanisms, and biological implications of translation termination–reinitiation

“…Reinitiation at a downstream ORF in the polycistronic viral mRNA of cauliflower mosaic virus is dependent on the viral-encoded "transactivator/viroplasmin" protein, which has been found to interact with the 60S subunit, eIF3, and a "reinitiation supporting protein" in the plant host (Park et al 2001;Thiébeauld et al 2009;Mancera-Martínez et al 2021).…”

Do plants drive translation reinitiation to dodge nonsense-mediated decay?

“…In plants, TOR is significantly implicated in translation reinitiation of a multitude of mRNAs that harbour uORFs within their leader regions. In plants, an active TOR relay can overcome uORF repression by promoting reinitiation after uORF translation via a series of phosphorylation events, such as phosphorylation of eIF3 subunit h (eIF3h; Schepetilnikov et al ., 2013 ), reinitiation supporting protein (RISP), and the 40S ribosomal protein S6 (rpS6; Mancera-Martínez et al ., 2021 ). In Arabidopsis, a C-terminal truncation of eIF3h has no major effect on the first initiation event but almost abolishes mRNA reinitiation capacity.…”

“…Indeed, h-less eIF3 can still interact with 40S and promote cap-dependent initiation events, indicating that eIF3h is a specific translation reinitiation factor in plants. Recent work also provides striking insight into the function of rpS6 in translation ( Mancera-Martínez et al ., 2021 ). Although rpS6 phosphorylation was not demonstrated to have a positive effect on global protein synthesis, its high phosphorylation status is important for reinitiation after uORF translation.…”

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs

“…Another important player in this process is the cellular protein RISP, which acts as a TAV partner in the binding of eIF3, 40S, and 60S subunits [ 236 ]. In the recent study by the same group [ 237 ], an interesting model has been proposed, according to which an elongated RISP molecule, interacting simultaneously with the eS6/RSP6 and eL24/RPL24 proteins, acts as a clamp, holding together the large and small ribosomal subunits. In this case, binding is regulated by phosphorylation of eS6 and RISP itself by the TOR-S6K1 signaling cascade.…”