Specificity and evolutionary conservation of the Escherichia coli RNA pyrophosphohydrolase RppH

Abstract

Bacterial RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5'-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5' end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5' terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.

Keywords: Nudix; Proteobacteria; RNA Degradation; RNA Modification; RNA Turnover; RNA-Protein Interaction; mRNA Decay.

FIGURE 1.

EcRppH substrates. The sequence and expected secondary structure of A8, A4, A3, A2, A1, A1+3, G8, and A8XL RNA are shown. Each bore a 5′-terminal triphosphate (ppp), a γ-³²P radiolabel (*) at the 5′ end, and a fluorescein label at the top of the first stem-loop. In each RNA name, the letter indicates the identity of the 5′-terminal nucleotide, and the numeral indicates the number of unpaired nucleotides at the 5′ end. Truncated derivatives of A8 (A4, A3, A2, A1) lacked 4–7 nucleotides from the 3′ boundary of the 5′-terminal single-stranded segment. G8, G4, G3, G2, G1, and G0 were identical to their A-series counterparts except for the presence of G instead of A at the 5′ end. A1+3 was the same as A1 except for three additional nucleotides at the 3′ end.

FIGURE 2.

Effect of the length of the 5′-terminal single-stranded segment on reactivity with EcRppH in vitro. A, representative gel images. In vitro transcribed A8 and A1 bearing a γ-³²P radiolabel and an internal fluorescein label were mixed with labeled A8XL and treated with purified EcRppH (8 nm), and the radioactivity (P-32) and fluorescence (Fluor) of each RNA were monitored as a function of time by gel electrophoresis. B and C, graphs. RppH-catalyzed phosphate removal from A8, A4, A3, A2, A1, and A1+3 or from G8, G4, G3, G2, G1, and G0 was monitored as in A and quantified by normalizing the radioactivity remaining in each RNA to the corresponding fluorescence intensity. Each time point is the average of two or more independent measurements. Error bars have been omitted to improve the legibility of the graph; instead, the standard error of each measurement is reported in supplemental Table 1.

FIGURE 3.

Effect of the sequence of the first three RNA nucleotides on reactivity with EcRppH in vitro. A, position 1. The reactivity of A4_AGAA and G4_GGAA was compared as in Fig. 2. The subscript in each RNA name indicates the sequence of the four unpaired nucleotides at the 5′ end. Consequently, A4_AGAA was equivalent to A4. B and C, position 2. The reactivity of A4_AGAA, A4_ACAA, and A4_AUAA and of G4_GGAA, G4_GAAA, G4_GCAA, and G4_GUAA was compared. Although both radioactivity and fluorescence were measured, only the former is shown in the gel images. To avoid modifying the second nucleotide, A4_AUAA and G4_GUAA were not labeled with fluorescein; instead, the fluorescence of fluorescein-labeled A8XL was used to normalize the data from each time point. The synthesis of A4_AAAA was not successful. D, position 3. The reactivity of A4_AGAA, A4_AGGA, A4_AGCA, and A4_AGUA was compared. To avoid modifying the third nucleotide, A4_AGUA was not labeled with fluorescein. The standard error of each measurement is reported in supplemental Table 1.

FIGURE 4.

Effect of the identity of the second RNA nucleotide on monophosphorylation in vivo. Total RNA was extracted from log-phase E. coli cells containing either wild-type yeiP mRNA (5′ AU) or a variant (yeiP-U2G) in which the second nucleotide was changed from U to G (5′ AG), and the phosphorylation state of each transcript was determined by PABLO. RNA samples that had first been treated in vitro with an excess of purified EcRppH (PPase) were analyzed in parallel so that the ligation yields of the fully monophosphorylated transcripts could be used as correction factors for calculating the percentage of yeiP and yeiP-U2G that was monophosphorylated. The steady-state ratios of monophosphorylated to triphosphorylated mRNA reported in the text (mean values and standard deviations) were each calculated from measurements made on three independent RNA preparations.

FIGURE 5.

RppH sequence alignment. A, alignment of EcRppH and BsRppH. The sequences were aligned by analysis with ClustalW. Asterisks mark amino acid residues that are identical. Residues that are conserved in virtually all bacterial orthologs of EcRppH or BsRppH are depicted as red or blue letters, respectively. B, alignment of proteobacterial RppH orthologs. Sequences are shown for RppH from E. coli, Legionella pneumophila, Burkholderia mallei, Azoarcus sp. strain BH72, Rhodobacter sphaeroides, Agrobacterium tumefaciens, Campylobacter jejuni, and Nautilia profundicola, except that the nonhomologous carboxyl-terminal tails of the enzymes from E. coli (ENTPKPQNASAYRRKRG), L. pneumophila (RRTPYGLKRKRGNQRA), B. mallei (QRTDKSRGPRAPRYPRVANGHAASEAPAAIDTSAVCSEVEPGANALDETPPRVSLRD), and Azoarcus sp. strain BH72 (KPAELPEGYRQGTASQA) have been omitted. Asterisks or colons mark amino acid residues that are identical or chemically similar, respectively. Residues that were mutated in the EcRppH variants characterized in Figs. 6 and 7 are depicted on a gray background. Numbers correspond to the sequence of EcRppH. C, evolutionary conservation of EcRppH and BsRppH orthologs. The dendrogram includes only bacterial phyla in which recognizable sequence homologs of EcRppH (p value ≤3 × 10⁻¹²) or BsRppH (p value ≤4 × 10⁻¹⁵) are common. The number of species within a class or order whose genomes were represented in the Blastp database and found to encode an ortholog of EcRppH (red) or BsRppH (blue) is indicated. Phylogenetic branching reflects RppH clades, as calculated by ClustalW2. Line lengths are arbitrary.

FIGURE 6.

Effect of mutations on the activity of EcRppH. A, mutation of Mg²⁺-coordinating glutamate residues. The catalytic activity of EcRppH bearing an alanine substitution for Glu-53, Glu-56, Glu-57, or Glu-120 was compared with that of wild-type EcRppH (WT) by monitoring phosphate removal from A4_AGAA, as in Fig. 2. B, mutation of residues that contact the base of the second RNA nucleotide. The catalytic activity of EcRppH bearing a mutation of Arg-27, Ser-32, Val-137, Phe-139, or Lys-140 was compared. The standard error of each measurement is reported in supplemental Table 1.

FIGURE 7.

Effect of mutations on the specificity of EcRppH. A, representative gel images. The substrate specificity of various EcRppH mutants was compared with that of wild-type EcRppH (WT) by monitoring phosphate removal from A4_AGAA and A4_ACAA, as in Fig. 2. To achieve greater balance in the reaction rates, four times more EcRppH was added to A4_ACAA than to A4_AGAA. Although both radioactivity and fluorescence were measured, only the former is shown in the gel images. B, graph. After normalizing radioactivity to fluorescence and then averaging data from multiple experiments, the ratio of triphosphate remaining on A4_AGAA versus A4_ACAA was calculated at each time point and plotted. The standard error of each measurement is reported in supplemental Table 1. A more negative slope indicates a greater preference for A4_AGAA over A4_ACAA. Because more EcRppH was added to A4_ACAA than to A4_AGAA, even an initial slope of 0 (horizontal line) signifies a residual preference for the latter substrate. Red zone, enhanced specificity mutants; green zone, diminished specificity mutants.

FIGURE 8.

Interaction of EcRppH with the second base of RNA substrates. A, structure of the nucleotide-binding pocket of EcRppH with a guanine ligand. The illustration is based on the EcRppH·2Mg·ppcpAGU structure reported in the accompanying article by Vasilyev and Serganov (20). The base of the second RNA nucleotide is blue; EcRppH side chains that contact the edge of the base (Ser-32 and Lys-140) are red; EcRppH side chains that contact the planar surfaces of the base (Arg-27, Val-137, and Phe-139) are pink; and water is green. Dashed lines represent hydrogen bonds. B, hypothetical structure of the nucleotide-binding pocket of EcRppH with a cytosine ligand.