Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• RNA-binding protein muš (RBPMS) is a key regulator of alternative splicing in differentiated smooth muscle cells (SMCs).
• RBPMS is transcriptionally downregulated during SMC dedifferentiation, suggesting post-translational modifications may acutely control its activity.

Purpose of the Study:

• To investigate the role of RBPMS post-translational modifications, specifically phosphorylation, in regulating its splicing activity.
• To identify the specific phosphorylation sites and the kinase responsible for RBPMS modification.

Main Methods:

• Analysis of phosphoproteomic datasets to identify phosphorylation sites on RBPMS.
• Creation and testing of phosphomimetic (T/E) and non-phosphorylatable (T/A) RBPMS mutants in cellular and in vitro assays.
• RNA binding affinity and oligomerization studies using biochemical methods.
• Nuclear Magnetic Resonance (NMR) analysis to elucidate the mechanism of inhibition.
• Kinase assays to identify the responsible kinase.

Main Results:

• Phosphorylation commonly occurs at Thr113 and Thr118 near the RRM domain.
• The phosphomimetic T/E mutant exhibited reduced splicing regulatory activity and RNA binding compared to the T/A mutant.
• NMR analysis suggested the T/E peptide acts as an RNA mimic, inhibiting RBPMS RNA binding.
• ERK2 was identified as the kinase responsible for RBPMS phosphorylation at Thr113 and Thr118.

Conclusions:

• Phosphorylation of RBPMS at Thr113/118 by ERK2 provides a mechanism for rapid, acute downregulation of its splicing activity.
• This post-translational modification allows for swift modulation of the SMC splicing program in response to external signals.
• This regulatory mechanism is crucial for processes like the vascular injury response and atherogenesis.