Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinformatics

Background:

• RNA-binding proteins (RBPs) are essential for cellular functions and feature intrinsically disordered regions.
• These disordered regions often contain repetitive sequences, including newly identified electronegative clusters (ENCs) rich in acidic residues and phosphorylation sites.

Purpose of the Study:

• To investigate the functional roles of ENCs in RBPs, focusing on protein stability, RNA-binding affinity, and specificity.
• To explore the impact of ENCs on the sequence specificity of RNA binding.

Main Methods:

• Bioinformatic analysis to identify and quantify repetitive sequences in RBPs.
• Biochemical assays using Nop15 (ribosomal biogenesis factor 15) as a model RBP.
• Engineering an ENC into Ser/Arg-rich splicing factor 3 and subsequent RNA Bind-n-Seq analysis.

Main Results:

• ENCs are abundant and longer than other known repetitive sequences in RBPs.
• The ENC in Nop15 increased protein stability and inhibited nonspecific RNA binding while minimally affecting specific binding.
• Engineered ENCs modulated RNA motif binding differently, with electrostatic interaction sites being more susceptible to inhibition.

Conclusions:

• ENCs play a significant role in regulating RNA binding, primarily through electrostatic interactions.
• The prevalence of ENCs in DNA-binding proteins and their underrepresentation in halophiles supports their role in modulating nucleic acid interactions.