Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative...

Area of Science:

• Molecular Biology
• RNA Metabolism
• Protein Phosphorylation

Background:

• Ser/Arg-rich (SR) proteins are nuclear phosphoproteins crucial for mRNA metabolism.
• Shuttling SR proteins associate with ribosomes and enhance mRNA translation.
• The phosphorylation state of SR proteins may influence their cytoplasmic functions.

Purpose of the Study:

• To investigate the role of SF2/ASF phosphorylation in cytoplasmic RNA processing.
• To determine the impact of RS domain phosphorylation on SF2/ASF binding to mRNA and translation.
• To identify critical domains of SF2/ASF involved in mRNA translation.

Main Methods:

• Analysis of endogenous cytoplasmic SF2/ASF phosphorylation state.
• Site-directed mutagenesis to mimic hypophosphorylated RS domain.
• In vivo and in vitro assays for SF2/ASF binding to mRNA and translation activity.
• Assessment of SF2/ASF RNA recognition motif 2 (RRM2) function.

Main Results:

• Cytoplasmic SF2/ASF associated with translation machinery is hypophosphorylated.
• Mutations mimicking hypophosphorylation increased SF2/ASF binding to mRNA and translation activity.
• The RS domain is not essential for SF2/ASF translation function, but RRM2 is critical.
• RS domain phosphorylation influences mRNA association, while RRM2 mediates protein-protein interactions.

Conclusions:

• RS-domain phosphorylation regulates SF2/ASF association with mRNA.
• RRM2 domain is crucial for SF2/ASF-mediated translation.
• Reversible protein phosphorylation differentially controls SR protein localization and activity.