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RNA Biol. 2020 Feb 12:1-14. doi: 10.1080/15476286.2020.1720376. [Epub ahead of print]

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

Author information

1
Department of Microbiology and Molecular Medicine, CMU, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
2
Protein Production Platform, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
3
Microbiology Unit, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland.

Abstract

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

KEYWORDS:

ATPase; DExH-box; HDX-MS; Pseudomonas aeruginosa; RNA helicase; RNA-binding proteins

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