Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

Area of Science:

• Molecular Biology
• Cell Biology
• RNA Biology

Background:

• Alternative splicing regulation is critical for adipogenesis, but poorly understood.
• Sam68 (RNA binding protein) is a known alternative splicing regulator.
• Sam68-deficient mice show adipogenesis defects linked to mTOR signaling.

Purpose of the Study:

• Investigate Sam68's role in adipogenesis.
• Identify alternative splicing imbalances in mTOR pathway components in Sam68-deficient cells.
• Determine Sam68's regulation of Rps6kb1 (ribosomal S6 kinase gene) isoform expression.

Main Methods:

• Cross-linking and immunoprecipitation (CLIP) assays.
• Minigene assays.
• Small interfering RNA (siRNA) mediated gene silencing.
• Ectopic expression studies in 3T3-L1 cells.

Main Results:

• Sam68 regulates Rps6kb1 isoform expression, specifically preventing Rps6kb1-002 and p31S6K1 protein production in wild-type adipocytes.
• Sam68 binds Rps6kb1 intron 6, inhibiting SRSF1-mediated splicing of Rps6kb1-002.
• p31S6K1 depletion partially rescues adipogenesis in Sam68-deficient cells.
• Ectopic p31S6K1 expression inhibits adipogenesis in wild-type cells.

Conclusions:

• Sam68 is required to suppress p31S6K1 expression for adipogenesis to proceed.
• p31S6K1 acts as an inhibitor of adipogenesis.
• Sam68's regulation of Rps6kb1 splicing is a key mechanism controlling adipogenesis.