hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its...

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Prion-like proteins form dynamic, multivalent assemblies and undergo liquid-liquid phase separation (LLPS) into membraneless organelles.
• Alternative splicing (AS) is a key mechanism regulating RNA-binding protein function by altering protein domains and assembly properties.
• Heterogeneous ribonucleoprotein D-like (hnRNPDL) is an RNA-processing protein with AS isoforms that differ in disordered domains, potentially influencing its assembly and splicing activity.

Purpose of the Study:

• To investigate how AS of hnRNPDL affects its phase separation, complex formation, nucleocytoplasmic shuttling, and amyloidogenicity.
• To determine the impact of disease-associated mutations in hnRNPDL on its assembly properties and protein solubility.
• To elucidate the molecular mechanism underlying limb-girdle muscular dystrophy 1G (LGMD1G) linked to hnRNPDL mutations.

Main Methods:

• Utilized in vitro and in vivo assays to study hnRNPDL phase separation and complex dynamics.
• Employed Drosophila models to assess the effects of hnRNPDL mutations on protein aggregation and solubility in muscle tissue.
• Analyzed the role of alternative splicing in modulating hnRNPDL assembly and function.

Main Results:

• Demonstrated that AS of hnRNPDL significantly controls its phase separation behavior.
• Showed that AS influences the size and dynamics of hnRNPDL nuclear complexes and its shuttling between the nucleus and cytoplasm.
• Revealed that disease-associated mutations (D378H/N) in hnRNPDL accelerate protein aggregation and reduce solubility in Drosophila muscle.

Conclusions:

• Alternative splicing is a critical regulator of hnRNPDL's biophysical properties, including phase separation and amyloid formation.
• hnRNPDL mutations linked to LGMD1G impair its assembly properties, leading to accelerated aggregation and reduced solubility.
• These findings suggest a genetic loss-of-function mechanism for LGMD1G, driven by aberrant hnRNPDL aggregation.