RNA ligases and RNA repair

RNA ligases and RNA repair

Share
Share

Overview of RNA repair There is an emerging appreciation of the existence of “RNA repair” pathways that rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful RNA breakage events. 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, unfolded protein response). RNA cleavage can occur either: (i) by a transesterification mechanism that yields 2’,3’-cyclic-PO4 and 5’-OH ends; or (ii) via a hydrolytic mechanism that leaves 3’-OH and 5’-PO4 ends. RNA repair enzymes capable of sealing 2’,3’-cyclic-PO4/5’-OH breaks or 3’-OH/5’-PO4 breaks are present in diverse taxa in all phylogenetic domains of life.     

ATP-dependent RNA ligases join 3’-OH and 5’-PO4 RNA termini via three nucleotidyl transfer steps similar to those of DNA ligases: (Step 1) ligase reacts with ATP to form a covalent ligase-(lysyl-Nz)–AMP intermediate plus pyrophosphate; (Step 2) AMP is transferred from ligase-adenylate to the 5’-PO4 RNA end to form an RNA–adenylate intermediate (AppRNA); and (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. In order for these RNA ligases to repair breaks formed by transesterification, the 2’,3’-cyclic-PO4 and 5’-OH ends must first be “healed”. End-healing entails hydrolysis of the 2’,3’-cyclic phosphate (by a phosphoesterase enzyme) to form a 3’-OH and phosphorylation of the 5’-OH (by a polynucleotide kinase enzyme) to form a 5’-PO4.

Phage T4 polynucleotide kinase/phosphatase (Pnkp) and RNA ligase 1 (Rnl1) are prototypal RNA healing and sealing enzymes. They function in vivo to repair a break in E. coli tRNALys triggered by phage-activation of a latent host-encoded anticodon nuclease PrrC. PrrC specifically incises tRNALys at a single site in the anticodon loop, 5’ of the wobble uridine, to generate a 2’,3’-cyclic-PO4//5’-OH break. If Pnkp and Rnl1 are not present, depletion of the pool of functional tRNALys by PrrC blocks phage protein synthesis and arrests the infection before it can spread. However, Pnkp and Rnl1 repair the broken tRNAs and thereby thwart the host defense mechanism. Pnkp remodels the ends of the broken tRNA as follows: (i) it converts the 2’,3’-cyclic-PO4 to a 3’-PO4 and then hydrolyzes the 3’-PO4 to a 3’-OH; and (ii) it transfers the gamma phosphate from ATP to the 5’-OH end to form a 5’-PO4. Rnl1 then joins the 3’-OH and 5’-PO4 RNA ends. We have probed in depth the structural basis for catalysis by T4 Pnkp and Rnl1 and the features of the PrrC anticodon nuclease that are required for “ribotoxicity” in vivo.

Fungal tRNA Splicing as a Paradigm of RNA Repair  – Intron-containing tRNAs are widespread in eukarya; the intron is usually located in the anticodon loop of the pre-tRNA and must be removed precisely for the tRNA to function in protein synthesis. tRNA splicing occurs in two stages: (i) intron excision and (ii) joining of the broken tRNA halves. A tRNA splicing endonuclease catalyzes dual incisions of the pre-tRNA at the exon-intron borders to yield 2’,3’-cyclic-PO4 and 5’-OH termini. The joining phase of tRNA splicing has been characterized best in fungi and plants, where a single multifunctional tRNA ligase enzyme (Trl1 in yeast; AtRNL in Arabidopsis) repairs the ends of the broken tRNA half-molecules. The healing and sealing steps of yeast/plant tRNA splicing involve distinctive intermediates and an additional repair step not seen in the phage T4 tRNA repair pathway. Trl1 and AtRNL perform three reactions: (i) the 2’,3’-cyclic-PO4 of the proximal tRNA half-molecule is hydrolyzed to a 3’-OH,2’-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5’-OH of the distal half-molecule is phosphorylated by a kinase; and (iii) the 3’-OH,2’-PO4 and 5’-PO4 ends are sealed by an ATP-dependent RNA ligase to form a spliced tRNA with an unconventional 2’-PO4, 3’-5’ phosphodiester at the splice junction. The final step of removing of the 2’-PO4 is performed by a separate enzyme, Tpt1.

Trl1 (827-aa) and AtRNL (1104-aa) are homologous proteins composed of separable healing and sealing enzyme modules. The C-terminal CPD module belongs to the “2H” phosphoesterase superfamily, named for the pair of HxT peptides that comprise the CPD active site. The central kinase module is a member of the P-loop phosphotransferase superfamily, named for a signature GxGK(S/T) motif that binds the NTP phosphate donor. The N-terminal ATP-dependent ligase domain belongs to the covalent nucleotidyltransferase enzyme superfamily that includes classic RNA/DNA ligases and mRNA capping enzymes. We’ve shown that: (i) each component catalytic module of tRNA ligase is essential for yeast cell growth; (ii) mutations in the active sites of the CPD, kinase, and ligase domains are lethal in vivo; and (iii) phage and plant tRNA repair systems are “portable” and can complement growth of a yeast trl1∆ null mutant. These findings empowered us to develop yeast trl1 mutants as genetic models for studies of phage, plant, and fungal tRNA repair systems and the identification and characterization of novel tRNA repair activities from eukarya, archaea, and bacteria.

During the last several years we made major strides in (i) elucidating the unique biochemical requirement for a 2’-PO4 in the ligation step of fungal/plant tRNA ligase; (ii) developing a kinetic mechanism for the healing and sealing steps; (iii) highlighting distinctive kinetic properties and NTP specificities of the kinase components of fungal versus plant tRNA ligase; and (iv) solving the crystal structure of the kinase domains of Candida tRNA ligase.

tRNA Ligase as Antifungal Drug Target – Fungal/plant tRNA ligases are essential agents of informational and stress response pathways involving the repair of tRNA breaks (during tRNA splicing) and mRNA breaks (during the unfolded protein response). The biochemical outcomes and end-specificities of the healing and sealing reactions they catalyze are unique compared to the tRNA repair systems elaborated by viruses, bacteria, and metazoans. Indeed, metazoan proteomes have no discernable homologs of the sealing domain of fungal/plant tRNA ligase. Metazoa rely for end-joining during tRNA splicing and the unfolded protein response on RtcB enzymes, a novel clade of RNA ligases co-discovered by my lab. RtcB is a GTP-dependent RNA ligase with no structural similarity to the classic ATP-dependent ligase superfamily. We’ve shown that RtcB splices 3’-PO4 and 5’-OH ends via a unique chemical mechanism entailing the formation of covalent RtcB-(histidinyl)-GMP and RNA3’pp5’G intermediates. There is no 5’ kinase end-healing step in the RtcB pathway of RNA repair. RtcB is conspicuously absent from the proteomes of pathogenic fungi. This scenario highlights tRNA ligase as a target for the discovery of new antifungals. 

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 have characterized Trl1 from the human fungal pathogens Aspergillus fumigatus, Coccidioides immitis, and Candida albicans. A. fumigatus causes invasive pulmonary disease in immunocompromised individuals, especially those with hematological malignancies or who have undergone bone marrow or solid organ transplantation. C. immitis is the agent of San Joaquin Valley Fever, a disease prevalent in the US desert Southwest region. C. albicans causes a spectrum of illnesses ranging from local infections of oral and genital mucosa to invasive systemic infections with significant morbidity and mortality in immunocompromised hosts. The Aspergillus, Coccidioides, and Candida Trl1 enzymes all strongly prefer GTP as the NTP phosphate donor for their 5’ kinase reactions. 

We recently solved a 2.2 Å crystal structure of the Candida Trl1 kinase domain in complex 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.

 

  1. Wang, L.K., and Shuman, S. (2001) Domain structure and mutational analysis of T4 polynucleotide kinase. J. Biol. Chem. 276, 26868-26874.
  2. Wang, L.K., and Shuman, S. (2002) Mutational analysis defines the 5’-kinase and 3’-phosphatase active sites of T4 polynucleotide kinase. Nucleic Acids. Res. 30, 1073-1080. 
  3. Wang, L.K., Lima, C.D., and Shuman, S. (2002) Structure and mechanism of T4 polynucleotide kinase – an RNA repair enzyme. EMBO J. 21, 3873-3880. 
  4. Wang, L.K., Ho, C.K., Pei, Y., and Shuman, S. (2003) Mutational analysis of bacteriophage T4 RNA ligase 1: different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction. J. Biol. Chem. 278, 29454-29462.
  5. Sawaya, R., Schwer, B., and Shuman, S. (2003) Genetic and biochemical analysis of the functional domains of yeast tRNA ligase. J. Biol. Chem. 278, 43298-43398.
  6. Schwer, B., Sawaya, R., Ho, C.K., and Shuman, S. (2004) Portability and fidelity of RNA-repair systems. Proc. Natl. Acad. Sci. USA 101, 2788-2793. 
  7. Ho, C.K., Wang, L.K., Lima, C.D., and Shuman, S. (2004) Structure and mechanism of RNA ligase. Structure 12, 327-339.
  8. Nandakumar, J., and Shuman, S. (2004) How an RNA ligase discriminates RNA damage versus DNA damage. Molecular Cell 16, 211-221.
  9. Nandakumar, J., Ho, C.K., Lima, C.D., and Shuman, S. (2004) RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J. Biol. Chem. 279, 31337-31347.
  10. Martins, A., and Shuman, S. (2004) An RNA ligase from Deinococcus radiodurans. J. Biol. Chem. 279, 50654-50661.
  11. Wang, L.K., and Shuman, S. (2005) Structure-function analysis of yeast tRNA ligase. RNA 11, 966-975. 
  12. Sawaya, R., Schwer, B., and Shuman, S. (2005) Structure-function analysis of the yeast NAD+-dependent tRNA 2’-phosphotransferase Tpt1. RNA 11, 107-113.
  13. Martins, A., and Shuman, S. (2005) An end-healing enzyme from Clostridium thermocellum with 5’ kinase, 2’,3’ phosphatase, and adenylyltransferase activities. RNA 11, 1271-1280. 
  14. Keppetipola, N., and Shuman, S. (2006) Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase. RNA 12, 73-82. 
  15. Keppetipola, N., and Shuman, S., (2006) Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase. J. Biol. Chem. 281, 19251-19259.
  16. Wang, L.K., Schwer, B., Englert, M., Beier, H., and Shuman, S. (2006) Structure-function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog. Nucleic Acids Res. 34, 517-527.
  17. Wang, L.K., Schwer, B., and Shuman, S. (2006) Structure-guided mutational analysis of T4 RNA ligase 1. RNA 12, 2126-2134. 
  18. Nandakumar, J., Shuman, S., and Lima, C.D. (2006) RNA ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward. Cell 127, 71-84.
  19. Wang, L.K., Nandakumar, J., Schwer, B., and Shuman, S. (2007) The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair. RNA 13, 1235-1244
  20. Zhu, H., Smith, P. Wang, L.K., and Shuman, S. (2007) Structure-function analysis of the 3’-phosphatase component of T4 polynucleotide kinase/phosphatase. Virology 366, 126-136. 
  21. Raymond, A., and Shuman, S. (2007) Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5’-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick. Nucleic Acids Res. 35, 839-849. 
  22. Keppetipola, N., and Shuman, S. (2007) Characterization of the 2’,3’ cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase. Nucleic Acids Res. 35, 7721-7732.
  23. Keppetipola, N., Nandakumar, J., and Shuman, S. (2007) Reprogramming the tRNA splicing activity of a bacterial RNA repair enzyme. Nucleic Acids Res. 35, 3624-3630. 
  24. Schwer, B., Aronova, A., Ramirez, A., Braun, P. and Shuman, S. (2008) Mammalian 2’,3’ cyclic nucleotide phosphodiesterase (CNP) can function as a tRNA splicing enzyme in vivo. RNA 14, 204-210. 
  25. Ramirez, A., Shuman, S., and Schwer, B. (2008) Human RNA 5’-kinase (hClp1) can function as a tRNA splicing enzyme in vivo. RNA 14, 1737-1745. 
  26. Jain, R., and Shuman, S. (2009) Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1. RNA 15, 923-931. 
  27. Keppetipola, N., Jain, R., Meineke, B., Diver, M., and Shuman, S. (2009) Structure-activity relationships in Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease. RNA 15, 1036-1044.
  28. Wang, L.K., and Shuman, S. (2010) Mutational analysis of the 5’-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase. Nucleic Acids Res. 38, 1304-1311. 
  29. Jain, R., and Shuman, S. (2010) Bacterial Hen1 is a 3’ terminal RNA ribose 2’O-methyltransferase component of a bacterial RNA repair cassette. RNA 16, 316-323. 
  30. Jain, R., and Shuman, S. (2011) Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3’ terminal RNA ribose 2’O-methyltransferase. RNA 17, 429-438. 
  31. Jain, R., Poulos, M.G., Gros, J., Chakravarty, A.K., and Shuman, S. (2011) Substrate specificity and mutational analysis of Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease. RNA 17, 1336-1343.
  32. Meineke, B., Schwer, B., Schaffrath, R., and Shuman, S. (2011) Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC. Nucleic Acids Res. 39, 687-700. 
  33. Meineke, B., and Shuman, S. (2012) Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair. RNA 18, 145-154. 
  34. Meineke, B., and Shuman, S. (2012) Structure-function relations in the NTPase domain of the antiviral tRNA ribotoxin Escherichia coli PrrC. Virology 427, 144-150. 
  35. Smith, P., Wang, L.K., Nair, P.A., and Shuman, S. (2012) The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family. Proc. Natl. Acad. Sci. USA 109, 2296-2301. 
  36. Wang, L.K., Das, U., Smith, P., and Shuman, S. (2012) Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system. RNA 18, 2277-2286. 
  37. Das, U., Wang, L.K., Smith, P. and Shuman, S. (2013) Structural and biochemical analysis of the phosphate donor specificity of the polynucleotide kinase component of the bacterial Pnkp•Hen1 RNA repair system. Biochemistry 52, 4734-4743. 
  38. Das, U., Wang, L.K., Smith, P., Jacewicz, A., and Shuman, S. (2013) Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5’-OH oligonucleotide and a product complex with GDP•Mg2+ and 5’-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition. Nucleic Acids Res. 42, 1152-1161. 
  39. Wang, L.K., Smith, P., and Shuman, S. (2013) Structure and mechanism of the 2’,3’ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system. Nucleic Acids Res. 41, 5864-5873. 
  40. Das, U., and Shuman, S. (2013) Mechanism of RNA 2’,3’-cyclic phosphate end-healing by T4 polynucleotide kinase-phosphatase. Nucleic Acids Res. 41, 355-365. 
  41. Chauleau, M., and Shuman, S. (2013) Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3’-OH base mispairs and damaged base lesions. RNA 19, 1840-1847. 
  42. Remus, B.S., and Shuman, S. (2013) A kinetic framework for tRNA ligase and enforcement of a 2’-phosphate requirement for ligation highlights the design logic of an RNA repair machine. RNA 19, 659-669. 
  43. Remus, B.S., and Shuman, S. (2014) Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases. RNA 20, 462-473. 
  44. Das, U., Wang, L.K., Smith, P., Munir A., and Shuman, S. (2014) Structures of bacterial polynucleotide kinase in a Michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5′-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5′-phosphorylated oligonucleotide. J. Bacteriol. 196, 4285-4292. 
  45. Schmier, B.J., and Shuman, S. (2014) Effects of 3’-OH and 5’-PO4 base mispairs and damaged base lesions on the fidelity of nick sealing by Deinococcus radiodurans RNA ligase. J. Bacteriol. 196, 1704-1712. 
  46. Chakravarty, A.K., Smith, P., Jalan, R., and Shuman, S. (2014) Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination. Cell Reports 7, 339-347. 
  47. Remus, B.S., Jacewicz, A., and Shuman, S. (2014) Structure and mechanism of E. coli RNA 2’,3’-cyclic phosphodiesterase. RNA 20, 1697-1705. 
  48. Unciuleac, M.C., and Shuman, S. (2015) Characterization of a novel eukaryal nick sealing RNA ligase from Naegleria gruberi. RNA 21, 824-832. 
  49. Unciuleac, M.C., Goldgur, Y., and Shuman, S. (2015) Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase. Proc. Natl. Acad. Sci. USA 112, 13868-13873. 
  50. Remus, B.S., Schwer, B., and Shuman, S. (2016) Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis. RNA 22, 1500-1509.
  51. Schmier, B.J., Chen, X., Wolin, S., and Shuman, S. (2017) Deletion of the rnl gene encoding a nick-sealing RNA ligase sensitizes Deinococcus radiodurans to ionizing radiation. Nucleic Acids Res. 45, 3812-3821. 
  52. Remus, B.S., Goldgur, Y., and Shuman, S. (2017) Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase. Nucleic Acids Res. 45, 12945-12953.