Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Area of Science:

• Molecular Biology
• Structural Biology
• Cell Biology

Background:

• RNA-binding protein with multiple splicing (RBPMS) is crucial for mRNA regulation and cellular processes like axon guidance and cancer progression.
• The RBPMS protein contains a single RNA recognition motif (RRM) domain responsible for RNA binding.

Purpose of the Study:

• To investigate the structure-function relationship of the RBPMS RRM domain.
• To understand the binding mechanism of RBPMS RRM to CAC-containing RNA.
• To elucidate the role of RBPMS dimerization and RNA-binding interfaces in cellular functions.

Main Methods:

• X-ray crystallography to determine the structure of RBPMS RRM bound to RNA.
• Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to map RNA binding sites.
• Size exclusion chromatography and isothermal titration calorimetry to assess binding affinity and dimerization.
• HEK293 cell transfections with RBPMS mutants to evaluate in vivo function.

Main Results:

• The RBPMS RRM domain forms a symmetrical dimer in its free state.
• Each monomer of the RBPMS RRM dimer binds sequence-specifically to CAC RNA segments.
• Structure-guided mutations disrupting dimerization or RNA binding decreased binding affinity and impaired RBPMS localization to stress granules in cells.

Conclusions:

• The RBPMS RRM domain's ability to dimerize and bind RNA sequence-specifically is critical for its function.
• Dimerization and RNA-binding interfaces are essential for RBPMS localization and activity in vivo.
• These findings provide structural insights into RBPMS-RNA complex formation and its biological implications.