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

Area of Science:

• Molecular Biology
• Plant Science
• Virology

Background:

• Reinitiation supporting protein (RISP) is crucial for translation initiation in plants.
• RISP interacts with ribosomal subunits and initiation factors.
• Target-of-rapamycin (TOR) kinase regulates RISP phosphorylation.

Purpose of the Study:

• To investigate the role of RISP phosphorylation at Ser267 in translation reinitiation.
• To elucidate the molecular mechanisms by which RISP phosphorylation affects translation.
• To determine the impact of RISP phosphorylation on viral replication.

Main Methods:

• In vitro binding assays
• Plant protoplast transient expression
• Analysis of RISP phosphorylation mutants (RISP-S267A and RISP-S267D)
• Generation and analysis of eS6-deficient plants and phosphomimic lines

Main Results:

• RISP phosphorylation at Ser267 by TOR dictates its interaction with eIF2 or eS6.
• Phosphorylation-deficient RISP (RISP-S267A) promotes translation initiation.
• Phosphorylation-mimic RISP (RISP-S267D) binds eS6 and facilitates 60S ribosomal subunit loading.
• eS6-deficient plants exhibit resistance to Cauliflower Mosaic Virus (CaMV) due to impaired reinitiation.
• Restoration of eS6 function in deficient plants rescues reinitiation capacity.

Conclusions:

• RISP phosphorylation at Ser267 is a key regulatory switch for translation reinitiation in plants.
• The phosphorylation status of RISP fine-tunes its interaction partners to control translation initiation and reinitiation.
• This mechanism is critical for both cellular translation and viral replication strategies.