Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria
RNA helicases—central enzymes in RNA metabolism— often feature intrinsically disordered regions (IDRs) that enable phase separation and complex interactions with other proteins and/or RNA molecules. IDRs are varied and fast evolving, which makes their function hard to predict. In the bacterial pathogen Pseudomonas aeruginosa, two non-redundant RNA helicases, RhlE1 and RhlE2, share a conserved REC catalytic core but have different C-terminal extensions (CTEs) composed of IDRs of diverse length and amino acid composition. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both CTEs facilitate RNA binding and phase separation in vitro, leading to the in vivo localization of proteins in clusters within the cytoplasm. However, the CTE of RhlE2 is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping CTEs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-CTERhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-CTERhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases. Overall design: PAO1 wild type and mutants were grown in twelve 12-cm square LB plates at 16°C with 0.5% arabinose. Samples from 6 plates were pooled and three replicates of each strain were therefore obtained. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Illumina). Then, libraries were prepared using the Illumina TruSeq stranded mRNA kit and validated on the Bioanalyzer 2100 (Agilent). Samples were sequenced using the Illumina HiSeq 2000, 150 bp double end read at Novogene.