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

Over 50% of RNA-binding protein regions are disordered and characterized by repetitive sequences. We found that the most dominant repetitive sequences are electronegative clusters (ENCs) containing acidic residues and/or phosphorylation sites. Despite their abundance, ENCs’ functions are largely unknown. We elucidated the regulatory functions of ENCs using ribosomal biogenesis factor Nop15 as a model. Nop15 has an ENC and an RNA-recognition motif that binds to ITS2 RNA. We found that Nop15's ENC increases protein stability, and inhibits nonspecific RNA binding, but minimally affects specific binding. The ENC performs these regulatory functions through intramolecular interactions. We proposed that ENCs are selected by evolution for suppressing nonspecific binding. Consistent with this proposal, ENCs are also overrepresented in DNA-binding proteins, but underrepresented in halophiles in which nonspecific binding is less problematic. Using the high-throughput RNA Bind-n-Seq method, we investigated how an engineered ENC modulates the sequence specificity of RNA binding. We found that the engineered ENC increases RNA-binding specificity by downplaying the role of electrostatic interactions. In summary, our work revealed a general regulatory mechanism of ENCs. Considering that the regulatory function of ENCs depends on their length, ENCs can be conveniently used for design of more stable and specific RNA-binding proteins.