Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates - PubMed (original) (raw)

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

Jérémie Piton et al. Proc Natl Acad Sci U S A. 2013.

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.

Keywords: 5′-processing; RNA decapping; RNA stability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Structure of BsRppH. (A) Cartoon view of BsRppH showing the classical Nudix domain in dark blue (β strands) and yellow (α helices). The N-terminal domain, not visible in the BdRppH structure and absent from the EcRppH sequence, is shown in red. Strands and helices are labeled as for the BdRppH structure, with additional N-terminal strands labeled as β(−1) and β(−2). (B) Topology of BsRppH. Strands of the classical Nudix domain are shown in dark blue, helices in yellow and the N-terminal extension in red. (C) Salt bridges (green dotted lines) and hydrogen bonds (gray dotted lines) stabilizing N-terminal domain (red). (D) Hydrophobic interactions (blue arc) between helix α3 and N-terminal domain (red).

Fig. 2.

Fig. 2.

The nucleotide binding pocket of BsRppH is in a different location to the B. bacteriovorus enzyme. Superposition of BsRppH (green) and BdRppH (gray) structures. GTP residues bound to each enzyme are labeled. The three Mg ions of the BdRppH enzyme are shown as orange spheres.

Fig. 3.

Fig. 3.

Nucleotide and phosphate binding by BsRppH. Surface plots of BsRppH showing omit maps (Fo-Fc) of electron density corresponding to (A) GTP, (B) pcp-ppGpG, with G1 in the nucleotide binding pocket, and (C) pcp-ppGpG, with G2 in the nucleotide binding pocket, at 2.0 σ above the mean. Metal ions are shown as orange spheres. (D) Metal ion coordination of phosphate residues in the active site of BsRppH. (E) Metal ion coordination of phosphate residues in the active site of BdRppH.

Fig. 4.

Fig. 4.

Chemical shift mapping of BsRppH bound to one, two, or three nucleotides. Residues showing chemical shift variations upon binding to (A) pcp-GMP (B) pcp-pGpG, and (C) pcp-pGpGpA. Key residues are labeled, and the locations of the metal ions and GTP binding pocket are indicated. Assigned residues are in green and unassigned residues in gray. For each mixture, the average chemical shift variation (δ) was calculated (0.025 ppm for A, 0.07 ppm for B, and 0.08 ppm for C). Residues showing chemical shift variations are shown in colors ranging from beige (≥2δ) to orange (≥4δ) to violet (≥6δ) to red (disappearance), with the strength of the shift indicated both by the color (see gradient) and thickness of the backbone ribbon.

Fig. 5.

Fig. 5.

BsRppH has a preference for guanosine in position 2. (A) Representative autoradiograms of BsRppH reactions in vitro. The portions of the gel corresponding to the full-length (FL) γ-32P-labeled RNA substrate and inorganic phosphate (Pi) product are shown. The sequences of the first four nucleotides of the 280-nt substrates are shown above the autoradiogram, with mutations underlined. Right angled triangles indicate direction of increasing enzyme concentration (0.1, 0.3, and 1 μM). Specific activities were calculated from the lowest concentration of enzyme giving a visible product (Pi). Disappearance of the substrate is not a good indicator of enzyme activity; at higher enzyme concentrations, RppH forms a visible complex with the substrate that has difficulty entering the gel. (B) Histogram showing quantification of three independent experiments similar to that shown in A with SE as shown.

Fig. 6.

Fig. 6.

Hypothetical model of BsRppH bound to a trinucleotide RNA. Electrostatic surface map of BsRppH bound to pppGGA. Positively charged surfaces are shown in blue, negatively charges surfaces in red. Metal ions are shown as orange spheres. Nucleotides are labeled according to their position relative to the 5′ end.

Comment in

References

    1. Condon C. Maturation and degradation of RNA in bacteria. Curr Opin Microbiol. 2007;10(3):271–278. - PubMed
    1. Condon C. RNA processing in bacteria. In: Schaechter M, editor. Encyclopedia of Microbiology. 3rd Ed, Vol 5. Oxford: Elsevier; 2009. pp. 395–408.
    1. Deana A, Celesnik H, Belasco JG. The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal. Nature. 2008;451(7176):355–358. - PubMed
    1. Richards J, et al. An RNA pyrophosphohydrolase triggers 5′-exonucleolytic degradation of mRNA in Bacillus subtilis. Mol Cell. 2011;43(6):940–949. - PMC - PubMed
    1. Mackie GA. Ribonuclease E is a 5′-end-dependent endonuclease. Nature. 1998;395(6703):720–723. - PubMed

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