Formation of Polyphasic RNP Granules by Intrinsically Disordered Qβ Coat Proteins and Hairpin-Containing RNA

Area of Science:

• Cell Biology
• Biotechnology
• Biophysics

Background:

• RNA-protein (RNP) granules are essential cellular components formed through phase separation.
• Intrinsically disordered regions (IDRs) in proteins are key for RNP granule assembly and function.
• Previous work demonstrated synthetic RNA can induce RNP granule formation.

Purpose of the Study:

• To engineer programmable RNP granules using a phage coat protein with an IDR.
• To investigate the role of RNA valency in controlling RNP granule phase behavior.
• To assess the potential for enhancing protein production within these engineered granules.

Main Methods:

• Utilized a phage coat protein with an intrinsically disordered region (IDR).
• Engineered synthetic RNA molecules with varying numbers of hairpin structures.
• Performed in vivo studies to observe phase separation and granule dissolution.
• Employed multiple assays to analyze RNA valency and phase behavior.
• Incorporated a blue fluorescent protein gene into RNA to measure protein titer.

Main Results:

• The phage coat protein undergoes phase separation in vivo.
• RNA molecules with hairpins can dissolve pre-formed protein granules.
• RNA valency dictates distinct phase behaviors, creating programmable RNP granules.
• Engineered RNA with a fluorescent protein gene demonstrated a phase-dependent increase in protein titer.

Conclusions:

• Synthetic RNA and phage coat proteins can create tunable, polyphasic RNP granules.
• RNA valency is a critical factor in programming RNP granule formation and function.
• This platform offers a novel approach for engineering cellular compartments with applications in biotechnology.