Characterization of a novel eukaryal nick-sealing RNA ligase from <i>Naegleria gruberi</i>

Unciuleac, Mihaela-Carmen; Shuman, Stewart

doi:10.1261/rna.049197.114

Cited by 14 publications

(20 citation statements)

References 29 publications

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“…Electron density showed that AMP is covalently linked to the motif I Lys170 side-chain in the active site of the NTase domain (Fig. S2), consistent with our biochemical evidence that at least half of the recombinant NgrRnl protein was NgrRnl-AMP (29). The adenosine nucleotide is in the syn conformation.…”

Section: Resultssupporting

confidence: 85%

“…The structure highlights direct contacts to the ATP γ-phosphate from the N domain of NgrRnl, via Arg4 and Lys121, and to the second Mn 2+ coordination complex via Asp53. The N domain contacts to ATP and Mn2 in the Michaelis complex aid in orienting the PP i leaving group during step 1 lysine adenylylation, and account for the severe decrement in step 1 activity when the N domain is deleted (29). ) 2 Michaelis complex.…”

Section: Resultsmentioning

confidence: 99%

“…Deinococcus radiodurans RNA ligase (DraRnl) and Naegleria gruberi RNA ligase (NgrRnl) represent a recently appreciated, though structurally uncharacterized, Rnl5 family of ATP-dependent RNA ligase (26)(27)(28)(29). DraRnl and NgrRnl are template-directed RNA ligases capable of sealing nicked duplexes in which the 3′-OH strand is RNA.…”

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confidence: 99%

“…Instead, they contain a defining N-terminal domain that is important for overall 3′-OH/5′-PO 4 nick sealing and ligase adenylylation (step 1), but dispensable for phosphodiester synthesis at a preadenylylated nick (step 3). DraRnl and NgrRnl prefer manganese as the metal cofactor for nick sealing, although either Mg 2+ or Mn 2+ can support the formation of the Rnl-AMP intermediate (26,29). Manganese exerts profound effects on Deinococcus resistance to high-dose ionizing radiation and oxidative stress (30,31).…”

mentioning

confidence: 99%

“…(N. gruberi is an avirulent cousin of the human pathogen Naegleria fowleri, known as the "brain-eating amoeba," which causes a devastating infection, primary amoebic meningoencephalitis.) We have purified and characterized recombinant NgrRnl, the exemplary eukaryal Rnl5 ligase (29). Here we report atomic structures of NgrRnl, captured at three discrete steps along the lysine-adenylylation reaction pathway.…”

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confidence: 99%

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Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Unciuleac

Goldgur

Shuman

2015

Proc. Natl. Acad. Sci. U.S.A.

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ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO 4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl- 2+ complexhighlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pK a of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β-and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.NζRNA repair | covalent nucleotidyltransferase | lysyl-AMP B iochemically diverse RNA repair systems rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful RNA breakage events (1-5). RNA breaks destined for repair are inflicted by sequence-specific or structure-specific endoribonucleases during physiological RNA processing (e.g., tRNA splicing; kinetoplast mRNA editing) and under conditions of cellular stress (e.g., virus infection, starvation, unfolded protein response). RNA cleavage can occur either: (i) by a transesterification mechanism (generally metal-independent) that yields 2′,3′-cyclic-PO 4 and 5′-OH ends; or (ii) via a hydrolytic mechanism (typically metaldependent) that leaves 3′-OH and 5′-PO 4 ends. RNA repair enzymes capable of sealing 2′,3′-cyclic-PO 4 /5′-OH breaks or 3′-OH/5′-PO 4 breaks are distributed widely in all phylogenetic domains of life.ATP-dependent RNA ligases join 3′-OH and 5′-PO 4 RNA termini via a series of three nucleotidyl transfer steps similar to those of DNA ligases (6). In step 1, RNA ligase reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate plus pyrophosphate. In step 2, AMP is transferred from ligase-adenylate to the 5′-PO 4 RNA end to form an RNA-adenylate intermediate, AppRNA. In step 3, ligase catalyzes attack by an RNA 3′-OH on the RNA-adenylate to seal the two ends via a phosphodiester bond and release AMP.The autoadenylylation reaction of RNA ligases is performed by a nucleotidyltransferase (NTase) domain that is conserved in ATP-dependent and NAD + -dependent DNA ligases and GTPdependent mRNA capping enzymes (6, 7). The NTase domain includes six peptide motifs (I, Ia, III, IIIa, IV, and V) that form the nucleotide-bin...

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Unciuleac

Goldgur

Shuman

2015

Proc. Natl. Acad. Sci. U.S.A.

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Diversity of circular RNAs and RNA ligases in archaeal cells

2019

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Chemoproteomic discovery of a human RNA ligase

et al. 2023

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RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5′-PO4 and 3′-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5′-PO4 and 3′-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. These data provide the groundwork for establishing a human RNA repair pathway.

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Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Cited by 14 publications

References 29 publications

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Diversity of circular RNAs and RNA ligases in archaeal cells

Chemoproteomic discovery of a human RNA ligase

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