Characterization of the tRNA ligases of pathogenic fungi <i>Aspergillus fumigatus</i> and <i>Coccidioides immitis</i>

Remus, Barbara S.; Schwer, Beate; Shuman, Stewart

doi:10.1261/rna.057455.116

Cited by 15 publications

(22 citation statements)

References 29 publications

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“…Polynucleotide kinases (PNKs) are enzymes that play fundamental roles in numerous cellular processes including the repair of DNA, as well as the processing, maturation, repair, and degradation of RNA (Weinfeld et al 2011;Das et al 2014;Dikfidan et al 2014;Remus et al 2016). PNKs target the 5 ′ -terminus of DNA and RNA substrates and catalyze the transfer of the γ-phosphate from nucleoside triphosphate (NTP) to the 5 ′ -hydroxyl terminus of the polynucleotide substrate.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Pillon

Sobhany

Stanley

2018

RNA

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Grc3 is an essential well-conserved eukaryotic polynucleotide kinase (PNK) that cooperates with the endoribonuclease Las1 to process the preribosomal RNA (rRNA). Aside from being dependent upon Las1 for coordinated kinase and nuclease function, little is known about Grc3 substrate specificity and the molecular mechanisms governing kinase activity. Here we characterize the kinase activity of Grc3 and identify key similarities and differences between Grc3 and other polynucleotide kinase family members. In contrast to other PNK family members, Grc3 has distinct substrate preference for RNA substrates in vitro. By disrupting conserved residues found at the Grc3 kinase active site, we identified specific residues required to support Grc3-directed Las1-mediated pre-rRNA cleavage in vitro and in vivo. The crosstalk between Grc3 and Las1 ensures the direct coupling of cleavage and phosphorylation during pre-rRNA processing. Taken together, our studies provide key insight into the polynucleotide kinase activity of the essential enzyme Grc3 and its molecular crosstalk with the endoribonuclease Las1.

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Section: Introductionmentioning

confidence: 99%

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Pillon

Sobhany

Stanley

2018

RNA

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“…Fungal tRNA ligase Trl1 is an essential agent in the repair of programmed tRNA and mRNA breaks with 2′,3′-cyclic phosphate and 5′-OH ends that are generated during tRNA splicing and the unfolded protein response ( 1 , 2 ). Trl1 performs three distinct RNA repair reactions catalyzed by three distinct protein domains: (i) the 2′,3′-cyclic phosphate (>p) end is hydrolyzed to a 3′-OH,2′-PO 4 by a C-terminal cyclic phosphodiesterase (CPD) module that belongs to the 2H phosphoesterase superfamily; (ii) the 5′-OH end is phosphorylated by a central NTP-dependent polynucleotide kinase module of the P-loop phosphotransferase superfamily and (iii) the 3′-OH,2′-PO 4 and 5′-PO 4 ends are sealed by an N-terminal ATP-dependent RNA ligase module, of the covalent nucleotidyltransferase superfamily, to form an unconventional 2′-PO 4 , 3′-5′ phosphodiester at the splice junction ( 3 – 9 ).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, there is no 5′ kinase step in the RtcB pathway of RNA repair. Whereas polynucleotide kinases are widely distributed in nature, the kinase domains of fungal Trl1 enzymes are unique in that they have a strong preference for GTP as the phosphate donor ( 5 , 8 , 9 , 19 ). This raises the prospect that the fungal kinase donor site could be targeted by a small molecule inhibitor that interacts with the guanine specificity determinant(s) of Trl1, but does not bind to the donor sites of the many other cellular P-loop phosphotransferases, which either have no NTP donor preference or prefer ATP as substrates.…”

Section: Introductionmentioning

confidence: 99%

“…To fortify the case for fungal tRNA ligase as a drug target, we need to understand the properties of Trl1 enzymes produced by fungi that cause human disease. To that end, we previously characterized Trl1 from the human fungal pathogens Aspergillus fumigatus and Coccidioides immitis ( 9 ). Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised individuals, especially those with hematological malignancies or who have undergone bone marrow or solid organ transplantation.…”

Section: Introductionmentioning

confidence: 99%

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Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase

Remus

Goldgur

Shuman

2017

Nucleic Acids Research

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Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3′-OH,2′-PO4 and 5′-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and are promising targets for antifungal drug discovery because their domain compositions and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. A distinctive feature of Trl1 is its preferential use of GTP as phosphate donor for the RNA kinase reaction. Here we report the 2.2 Å crystal structure of the kinase domain of Trl1 from the fungal pathogen Candida albicans with GDP and Mg2+ in the active site. The P-loop phosphotransferase fold of the kinase is embellished by a unique ‘G-loop’ element that accounts for guanine nucleotide specificity. Mutations of amino acids that contact the guanine nucleobase efface kinase activity in vitro and Trl1 function in vivo. Our findings fortify the case for the Trl1 kinase as an antifungal target.

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“…Whereas T4 Pnkp, Clostridium thermocellum Pnkp, and the kinase of the plant tRNA ligase Arabidopsis thaliana RNA ligase (AtRNL) use any NTP as a phosphate donor (13,23), other Pnks are more fastidious. The Pnks of fungal tRNA ligases have a strong preference for GTP (24,46,47). In contrast, the human RNA kinase Clp1 favors ATP over GTP (48).…”

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confidence: 99%

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

Munir

Shuman

2017

J Bacteriol

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5=-and 3=-end-healing reactions are key steps in nucleic acid break repair in which 5=-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3=-PO 4 or 2=,3=-cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5=-PO 4 and 3=-OH termini required for sealing by classic polynucleotide ligases. Endhealing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2=,3=-phosphoesterase HD domain and a C-terminal 5=-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5=-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2=,3=-cyclic phosphate, RNA 3=-phosphate, RNA 2=-phosphate, and DNA 3=-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCEThe present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5=-and 3=-end-healing enzymes that phosphorylate 5=-OH termini and dephosphorylate 2=,3=-cyclic-PO 4 , 3=-PO 4 , and 2=-PO 4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. KEYWORDS 3= phosphatase, nucleic acid repair, polynucleotide kinase P olynucleotide kinases (Pnks) are a widely distributed class of cellular and virusencoded nucleic acid repair enzymes that convert 5=-OH termini into 5=-PO 4 ends that can be sealed by RNA or DNA ligases. Pnks are members of the P-loop phosphotransferase superfamily; they catalyze metal-dependent transfer of the ␥ phosphate of a nucleoside triphosphate (NTP) donor to a 5=-OH polynucleotide acceptor. In many repair systems, a Pnk enzyme is fused in a modular fashion to one or more other repair enzymes within a single multifunctional polypeptide.For example, bacteriophage T4 encodes a bifunctional 5=-OH polynucleotide kinase-3=-phosphatase (Pnkp) consisting of an N-terminal Pnk domain fused to a C-terminal

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Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

Cited by 15 publications

References 29 publications

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

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