title: Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
abstract: The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.
authors.affiliation: Centre National de la Recherche Scientifique (CNRS), Unité Propre de Recherche 9073 (affiliated with Université Paris Diderot, Sorbonne Paris Cité) Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Piton
authors.forename: Valéry
authors.initials: V
authors.lastname: Larue
authors.forename: Yann
authors.initials: Y
authors.lastname: Thillier
authors.forename: Audrey
authors.initials: A
authors.lastname: Dorléans
authors.forename: Olivier
authors.initials: O
authors.lastname: Pellegrini
authors.forename: Inés
authors.initials: I
authors.lastname: Li de la Sierra-Gallay
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Vasseur
authors.forename: Françoise
authors.initials: F
authors.lastname: Debart
authors.forename: Carine
authors.initials: C
authors.lastname: Tisné
authors.forename: Ciarán
authors.initials: C
authors.lastname: Condon
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D006151:Guanosine / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D020871:RNA Stability / Q000235:genetics / Q000502:physiology*; D013045:Species Specificity; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
chemical_list: D006151:Guanosine; D006160:Guanosine Triphosphate; D009841:Oligonucleotides; D011755:Pyrophosphatases; D012315:RNA Caps; D000096964:Nudix Hydrolases
keywords: 5′-processing; RNA decapping; RNA stability