Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway

Abstract

Escherichia coli RtcB is a founding member of a family of manganese-dependent RNA repair enzymes that join RNA 2=,3=-cyclic phosphate (RNA>p) or RNA 3=-phosphate (RNAp) ends to 5=-OH RNA ( HO RNA) ends in a multistep pathway whereby RtcB (i) hydrolyzes RNA>p to RNAp, (ii) transfers GMP from GTP to RNAp to form to RNAppG, and (iii) directs the attack of 5=-OH on RNAppG to form a 3=-5= phosphodiester splice junction. The crystal structure of the homologous archaeal RtcB enzyme revealed an active site with two closely spaced manganese ions, Mn1 and Mn2, that interact with the GTP phosphates. By studying the reactions of wild-type E. coli RtcB and RtcB alanine mutants with 3=-phosphate-, 2=,3=-cyclic phosphate-, and 3=-ppG-terminated substrates, we found that enzymic constituents of the two metal coordination complexes (Cys78, His185, and His281 for Mn1 and Asp75, Cys78, and His168 for Mn2 in E. coli RtcB) play distinct catalytic roles. For example, whereas the C78A mutation abolished all steps assayed, the D75A mutation allowed cyclic phosphodiester hydrolysis but crippled 3=-phosphate guanylylation, and the H281A mutant was impaired in overall HO RNAp and HO RNA>p ligation but was able to seal a preguanylylated substrate. The archaeal counterpart of E. coli RtcB Arg189 coordinates a sulfate anion construed to mimic the position of an RNA phosphate. We propose that Arg189 coordinates a phosphodiester at the 5=-OH end, based on our findings that the R189A mutation slowed the step of RNAppG/ HO RNA sealing by a factor of 200 compared to that with wild-type RtcB while decreasing the rate of RNAppG formation by only 3-fold. IMPORTANCERtcB enzymes comprise a widely distributed family of manganese-and GTP-dependent RNA repair enzymes that ligate 2=,3=-cyclic phosphate ends to 5=-OH ends via RNA 3=-phosphate and RNA(3=)pp(5=)G intermediates. The RtcB active site includes two adjacent manganese ions that engage the GTP phosphates. Alanine scanning of Escherichia coli RtcB reveals distinct contributions of metal-binding residues Cys78, Asp75, and His281 at different steps of the RtcB pathway. The RNA contacts of RtcB are uncharted. Mutagenesis implicates Arg189 in engaging the 5=-OH RNA end. Escherichia coli RtcB exemplifies a novel family of RNA ligases implicated in tRNA splicing and RNA repair (1-7). Unlike classic RNA and DNA ligases, which join 3=-OH and 5=-phosphate ends, RtcB seals broken RNAs with 5=-OH ( HO RNA) and either 2=,3=-cyclic phosphate (RNAϾp) or 3=-phosphate (RNAp) ends. E. coli RtcB executes a multistep ligation pathway (5-7) entailing (i) reaction of the enzyme with GTP to form a covalent RtcB-(His337-N)-GMP intermediate, (ii) hydrolysis of RNAϾp to RNAp, (iii) transfer of guanylate from His337 to the polynucleotide 3=-phosphate to form a polynucleotide-(3=)pp(5=)G intermediate, and (iv) attack of a 5=-OH on the ϪNppG end to form the splice junction and liberate GMP (Fig. 1). The E. coli RtcB reaction pathway requires manganese as a divalent cation cofactor. The catalytic repertoire of E. coli ...

Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

“…We found that RtcB-mediated ligations were more efficient than ligations performed by endogenous ligases in HeLa cells. We also confirmed that the multistep RtcB mechanism proceeded via a terminal monophosphate intermediate [29][30][31][32]35,36]. This implies that 3 0 -phosphorylated miRNAs might also function as probes for the new CLIP method using RtcB.…”

“…5B,C). Together with the appearance of a signal (peak#2) corresponding to the respective 3′‐phosphate intermediate, these data provide further evidence for the multistep pathway that proceeds with hydrolysis of the cyclic phosphate followed by ligation of the 3′‐phosphate intermediate [29–32,35,36]. The data were confirmed in a second experiment in which the excess Mn 2+ was not depleted prior to LC‐MS analysis (data not shown).…”

“…On the other hand, a series of mechanistic studies on recombinant RtcB rather indicated a multistep process in which the cyclic phosphate is first opened to a 3 0 -monophosphate, followed by activation as a GMP-adduct and ligation from attack of a 5 0 -hydroxyl group (Fig. 5A) [29][30][31][32]35,36]. To clarify how this unusual ligation mechanism proceeded with the miRNA>p's in this study, we performed an isotope-labeling study in which RtcB-dependent cyclization of a stem-loop RNA derived from a miRNA precursor (pre-20>p) was performed in H 2…”

“…Direct ligation between RNA 5′‐OH and 2′, 3′‐cyclic phosphate termini has been suggested in HeLa lysate [19,37,38]. For bacterial RtcB, hydrolysis of the 2′, 3′‐cycP to a 3′‐P terminus and subsequent ligation of the 3′‐P terminus has been suggested [29–36]. (B) RtcB‐mediated cyclization of the stem‐loop RNApre‐20>pin H 2 16 O and (C) H 2 18 O. RtcB ligation was performed at 37 °C for 1 h in the presence of 50 m m Tris/HCl (pH = 8), 1 m m MnCl 2 , 0.1 m m GTP, 0.75 µ m RtcB and 0.5 µ m pre‐20>p. Ligation was quenched through the addition of Na 2 EDTA to a final concentration of 50 m m .…”

“…Cyclic phosphate species and their enzyme-mediated ligation reactions are well known in the field of tRNA splicing [19,20] and are postulated to have played roles in the 'RNA world' hypothesis [21][22][23][24][25]. Whereas in conventional ligation mechanisms, an activated 5 0 -phosphate group of an RNA is attacked by a 3 0 -hydroxyl group of a second ribonucleotide [26][27][28], RNA>p participate in an unusual mechanism of ligation that was first observed in HeLa cell lysates and involves the attack of a 5 0 -hydroxyl RNA [19,[29][30][31][32][33][34][35][36][37][38][39]. In mammalian cells, it occurs preferentially with RNA>p, but also with 3 0 -phosphate RNAs that are first cyclized by 3 0 -phosphate cyclase [19,[37][38][39].…”