Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• RNA helicases are crucial for RNA metabolism, often possessing intrinsically disordered regions (IDRs) that facilitate complex interactions and phase separation.
• Pseudomonas aeruginosa utilizes two non-redundant RNA helicases, RhlE1 and RhlE2, which share a catalytic core but differ in their C-terminal IDRs.

Purpose of the Study:

• To investigate how variations in intrinsically disordered regions (IDRs) between RhlE1 and RhlE2 RNA helicases confer functional specificity.
• To elucidate the roles of IDRs in RNA binding, phase separation, enzymatic activity, and interactions with other cellular factors.

Main Methods:

• Construction and analysis of chimeric RNA helicases with swapped IDRs.
• Biochemical assays to assess RNA unwinding activity.
• Analysis of protein-protein interactions, specifically with RNase E.
• Assessment of bacterial growth under varying temperature conditions.
• Localization studies of RNA helicases within the cytoplasm.

Main Results:

• Both RhlE1 and RhlE2 IDRs promote RNA binding and cytoplasmic phase separation.
• The RhlE2 IDR enhances RNA unwinding activity and phase separation more effectively than the RhlE1 IDR.
• The RhlE2 IDR mediates interaction with the RNase E endonuclease.
• Chimeric helicases exhibit distinct biochemical and functional properties compared to native forms.
• The RECRhlE1-IDRRhlE2 chimera improved cold growth and interacted with RNase E.
• The RECRhlE2-IDRRhlE1 chimera impaired bacterial growth at low temperatures, linked to aberrant RNA droplets.

Conclusions:

• Intrinsically disordered regions (IDRs) are key determinants of functional specificity in RNA helicases.
• IDRs modulate both the catalytic core activity and the subcellular localization of RNA helicases through phase separation.
• Understanding IDR diversity is essential for deciphering the functional differentiation of RNA helicases in bacteria like Pseudomonas aeruginosa.