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Analysis of orthologous groups reveals archease and DDX1 as tRNA splicing factors

Abstract

RNA ligases have essential roles in many cellular processes in eukaryotes, archaea and bacteria, including in RNA repair1,2 and stress-induced splicing of messenger RNA3. In archaea and eukaryotes, RNA ligases also have a role in transfer RNA splicing to generate functional tRNAs required for protein synthesis4,5,6,7. We recently identified the human tRNA splicing ligase, a multimeric protein complex with RTCB (also known as HSPC117, C22orf28, FAAP and D10Wsu52e) as the essential subunit8. The functions of the additional complex components ASW (also known as C2orf49), CGI-99 (also known as C14orf166), FAM98B and the DEAD-box helicase DDX1 in the context of RNA ligation have remained unclear. Taking advantage of clusters of eukaryotic orthologous groups, here we find that archease (ARCH; also known as ZBTB8OS), a protein of unknown function, is required for full activity of the human tRNA ligase complex and, in cooperation with DDX1, facilitates the formation of an RTCB–guanylate intermediate central to mammalian RNA ligation. Our findings define a role for DDX1 in the context of the human tRNA ligase complex and suggest that the widespread co-occurrence of archease and RtcB proteins implies evolutionary conservation of their functional interplay.

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Figure 1: Archease facilitates multiple enzymatic turnover of RTCB.
Figure 2: Archease co-purifies with human RTCB upon in vivo crosslink.
Figure 3: Archease cooperates with DDX1 to guanylate RTCB in vitro.
Figure 4: tRNA maturation relies on RTCB, archease and DDX1 in vitro and in living cells.

References

  1. Chan, C. M., Zhou, C. & Huang, R. H. Reconstituting bacterial RNA repair and modification in vitro. Science 326, 247 (2009)

    ADS  CAS  Article  Google Scholar 

  2. Nandakumar, J., Schwer, B., Schaffrath, R. & Shuman, S. RNA repair: an antidote to cytotoxic eukaryal RNA damage. Mol. Cell 31, 278–286 (2008)

    CAS  Article  Google Scholar 

  3. Sidrauski, C., Cox, J. S. & Walter, P. tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell 87, 405–413 (1996)

    CAS  Article  Google Scholar 

  4. Filipowicz, W. & Shatkin, A. J. Origin of splice junction phosphate in tRNAs processed by HeLa cell extract. Cell 32, 547–557 (1983)

    CAS  Article  Google Scholar 

  5. Konarska, M., Filipowicz, W. & Gross, H. J. RNA ligation via 2′-phosphomonoester, 3′5′-phosphodiester linkage: requirement of 2′,3′-cyclic phosphate termini and involvement of a 5′-hydroxyl polynucleotide kinase. Proc. Natl Acad. Sci. USA 79, 1474–1478 (1982)

    ADS  CAS  Article  Google Scholar 

  6. Laski, F. A., Fire, A. Z., RajBhandary, U. L. & Sharp, P. A. Characterization of tRNA precursor splicing in mammalian extracts. J. Biol. Chem. 258, 11974–11980 (1983)

    CAS  Article  Google Scholar 

  7. Zofallova, L., Guo, Y. & Gupta, R. Junction phosphate is derived from the precursor in the tRNA spliced by the archaeon Haloferax volcanii cell extract. RNA 6, 1019–1030 (2000)

    CAS  Article  Google Scholar 

  8. Popow, J. et al. HSPC117 is the essential subunit of a human tRNA splicing ligase complex. Science 331, 760–764 (2011)

    ADS  CAS  Article  Google Scholar 

  9. Popow, J., Schleiffer, A. & Martinez, J. Diversity and roles of (t)RNA ligases. Cell. Mol. Life Sci. 69, 2657–2670 (2012)

    CAS  Article  Google Scholar 

  10. Englert, M. & Beier, H. Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins. Nucleic Acids Res. 33, 388–399 (2005)

    CAS  Article  Google Scholar 

  11. Konarska, M., Filipowicz, W., Domdey, H. & Gross, H. J. Formation of a 2′-phosphomonoester, 3′,5′-phosphodiester linkage by a novel RNA ligase in wheat germ. Nature 293, 112–116 (1981)

    ADS  CAS  Article  Google Scholar 

  12. Phizicky, E. M., Consaul, S. A., Nehrke, K. W. & Abelson, J. Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. J. Biol. Chem. 267, 4577–4582 (1992)

    CAS  Article  Google Scholar 

  13. Koonin, E. V. et al. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes. Genome Biol. 5, R7 (2004)

    Article  Google Scholar 

  14. Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41 (2003)

    Article  Google Scholar 

  15. Canaves, J. M. Predicted role for the archease protein family based on structural and sequence analysis of TM1083 and MTH1598, two proteins structurally characterized through structural genomics efforts. Proteins 56, 19–27 (2004)

    CAS  Article  Google Scholar 

  16. Auxilien, S., El Khadali, F., Rasmussen, A., Douthwaite, S. & Grosjean, H. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase. J. Biol. Chem. 282, 18711–18721 (2007)

    CAS  Article  Google Scholar 

  17. Brzezicha, B. et al. Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA Leu (CAA). Nucleic Acids Res. 34, 6034–6043 (2006)

    CAS  Article  Google Scholar 

  18. Paushkin, S. V., Patel, M., Furia, B. S., Peltz, S. W. & Trotta, C. R. Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3′ end formation. Cell 117, 311–321 (2004)

    CAS  Article  Google Scholar 

  19. Chakravarty, A. K., Subbotin, R., Chait, B. T. & Shuman, S. RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. Proc. Natl Acad. Sci. USA 109, 6072–6077 (2012)

    ADS  CAS  Article  Google Scholar 

  20. Tanaka, N., Chakravarty, A. K., Maughan, B. & Shuman, S. A novel mechanism of RNA repair by RtcB via sequential 2′,3′-cyclic phosphodiesterase and 3′-phosphate/5′-hydroxyl ligation reactions. J. Biol. Chem. 286, 43134–43143 (2011)

    CAS  Article  Google Scholar 

  21. Pause, A. & Sonenberg, N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 11, 2643–2654 (1992)

    CAS  Article  Google Scholar 

  22. Perkins, K. K., Furneaux, H. & Hurwitz, J. Isolation and characterization of an RNA ligase from HeLa cells. Proc. Natl Acad. Sci. USA 82, 684–688 (1985)

    ADS  CAS  Article  Google Scholar 

  23. Godbout, R., Packer, M. & Bie, W. Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines. J. Biol. Chem. 273, 21161–21168 (1998)

    CAS  Article  Google Scholar 

  24. Kanai, Y., Dohmae, N. & Hirokawa, N. Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43, 513–525 (2004)

    CAS  Article  Google Scholar 

  25. Cao, D., Haussecker, D., Huang, Y. & Kay, M. A. Combined proteomic–RNAi screen for host factors involved in human hepatitis delta virus replication. RNA 15, 1971–1979 (2009)

    CAS  Article  Google Scholar 

  26. Edgcomb, S. P. et al. DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication. J. Mol. Biol. 415, 61–74 (2012)

    CAS  Article  Google Scholar 

  27. Zhang, Z. et al. DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells. Immunity 34, 866–878 (2011)

    CAS  Article  Google Scholar 

  28. Desai, K. K., Cheng, C. L., Bingman, C. A., Phillips, G. N., Jr & Raines, R. T. A tRNA splicing operon: archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation. Nucleic Acids Res. (2014)

  29. Dandekar, T., Snel, B., Huynen, M. & Bork, P. Conservation of gene order: a fingerprint of proteins that physically interact. Trends Biochem. Sci. 23, 324–328 (1998)

    CAS  Article  Google Scholar 

  30. Ciria, R., Abreu-Goodger, C., Morett, E. & Merino, E. GeConT: gene context analysis. Bioinformatics 20, 2307–2308 (2004)

    CAS  Article  Google Scholar 

  31. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

    CAS  Article  Google Scholar 

  32. Katoh, K. & Toh, H. Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinform. 9, 286–298 (2008)

    CAS  Article  Google Scholar 

  33. Thompson, J. D., Gibson, T. J. & Higgins, D. G. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinformatics Chapter 2, Unit 2.3. (2002)

  34. Yee, A. et al. An NMR approach to structural proteomics. Proc. Natl Acad. Sci. USA 99, 1825–1830 (2002)

    ADS  CAS  Article  Google Scholar 

  35. Pei, J. & Grishin, N. V. AL2CO: calculation of positional conservation in a protein sequence alignment. Bioinformatics 17, 700–712 (2001)

    CAS  Article  Google Scholar 

  36. van de Wetering, M. et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 (2003)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to thank S. Ameres, B. Beckmann, R. Hartmann, M. Hentze, M. Jantsch, B. Klaus, B. Mair, A. F. Nielsen, J.-M. Peters, T. Rapoport, R. Schroeder and S. Weitzer for help and advice, S. Bandini for the preparation of Flag–TSEN2 cell lines and critical discussions and J. Dammann and T. Lendl for technical assistance. This work has been funded by the Fonds zur Förderung der wissenschaftlichen Forschung (P24687), the GEN-AU 3 research programme (820982 Non-coding RNAs) (J.P.) and the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (J.M.).

Author information

Authors and Affiliations

Authors

Contributions

J.P. designed and carried out experiments and wrote the manuscript, J.J. designed and carried out experiments and contributed to writing the manuscript, A.S. performed bioinformatic analysis of archease and contributed to writing the manuscript, J.M. designed the experiments and contributed to writing the manuscript.

Corresponding author

Correspondence to Javier Martinez.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Kinetic characterization of Flag–RTCB.

a, SDS–PAGE (Coomassie blue) of Flag–RTCB affinity purified from HEK293 cells. b, SDS–PAGE of a 10 μl aliquot of Flag–RTCB and comparison with a dilution series of bovine serum albumin (BSA). Flag–RTCB concentrations were between 40 and 80 ng per 10 μl, corresponding to an approximate concentration of 100 nM. c, Fit of time course data depicted in Fig. 1c to an exponential one phase association model restricting the bottom value to zero using the software GraphPad. Obtained plateau values were plotted against the concentration of Flag–RTCB. The linear relation of plateau product concentrations with the amount of Flag–RTCB suggests that human RNA ligase catalyses a limited number of substrate turnovers (mean ± standard error of the mean (s.e.m.), N = 3). d, Estimation of initial rates of ligation from time course data in Fig. 1c (mean ± s.d., N = 3). e, Reaction rates derived by linear regression from the data depicted in Fig. 1c were plotted against the concentration of Flag–RTCB (mean ± s.d., N = 3).

Extended Data Figure 2 Archease and RTCB proteins share their phyletic distribution in eukaryotic model genomes.

a, Whereas most organisms appear to rely on both 5′–3′ and 3′–5′ RNA ligase mechanisms, the genomes of the vast majority of plants and fungi (as exemplified by the model organisms Saccharomyces cerevisiae, Schizosaccharomyces pombe and Arabidopsis thaliana) do not encode for RtcB proteins known to catalyse 3′–5′ RNA ligation. b, The table lists protein families exhibiting the same phyletic pattern as RtcB proteins (ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kog). Archease is the only protein of unknown function represented in this group of KOGs. Plus sign indicates the presence of a protein assigned to a KOG in a given model organism, whereas minus sign indicates its apparent absence.

Extended Data Figure 3 Dose–response curve of stimulation of Flag–RTCB by recombinant archease.

a, SDS–PAGE analysis of purified recombinant wild-type (WT) and mutant (D39A, K144A) hexahistidine-tagged human archease. b, Dose–response curve of stimulation of the ligase activity (225 nM substrate) of Flag–RTCB (22.5 nM) by archease (40 nM to 10.2 µM). The rates of formation of Pconcat (mean ± s.d., N = 3) were plotted versus the respective concentration of archease. Affinity-purified Flag–RTCB required relatively high concentrations of recombinant archease (approximately 1 μM) for half-maximal activation of its RNA ligase activity. c, A master reaction was assembled as described in Methods (30 nM Flag–RTCB, 300 nM substrate), incubated at 37 °C for 90 min (to reach the plateau phase) and transferred to ice. 4.5 μl aliquots of this pre-incubation were mixed with 1.5 μl of recombinant archease solution to final concentrations of 40 nM to 10.25 µM and equilibrated for 4 min at 37 °C. The concentrations of all cofactors present in the reaction mixture were adjusted accordingly. Aliquots of the reaction were quenched at 4, 5.5, 7 and 8.5 min and analysed by denaturing electrophoresis. Pilot experiments were carried out before to ensure near-linear behaviour of the reactions at the chosen conditions. Reaction rates were determined by linear regression and are stated as slopes (nM s−1) ± s.e.m. (N = 3).

Extended Data Figure 4 Archease is highly conserved from bacteria and archaea to metazoa.

a, Alignment of archease proteins from various prokaryotic and eukaryotic organisms. Positions of residues examined in this study are indicated above. Residues marked by an asterisk have recently been implicated in metal binding in the structure of an archease from Pyrococcus horikoshii28. b, Archease residues examined by mutagenesis (D39, K144 and T147) are highly conserved and positioned in close proximity on the surface of the protein as suggested by the NMR structure of an archease from Methanobacterium thermoautotrophicum (Protein Data Bank accession 1JW3). Colours indicate conservation (yellow indicates the highest and blue indicates the lowest degree of conservation).

Extended Data Figure 5 Affinity-purified Flag–archease associates with RTCB.

a, Reducing (2 M β-mercaptoethanol) and non-reducing western blot of Flag–archease purified from HEK293 cells in the presence or absence of DSP. b, Western blot of Flag control eluates prepared from non-transfected HEK293 cells treated with DSP or DMSO. c, tRNA cleavage assay (100 nM substrate) of Flag–TSEN2 (10 nM) affinity purified from HEK293 cells in the presence or absence of archease (7.5 μM). d, Quantification of data shown in c by phosphorimaging. e, SPR sensorgram overlay demonstrating concentration-dependent binding of archease to RTCB. f, Mathematical sensorgram fitting to a Langmuir 1:1 interaction model (using Flag–RTCB as analyte) at the indicated concentrations. Data are displayed as subtractive curves against an amine-activated reference surface. g, Kinetic constants (kon, koff, Kd) for archease and Flag–RTCB complexes of hexahistidine-tagged wild-type (WT) and mutant (D39A) archease in the absence of nucleotide triphosphate cofactors, and the presence of GTP and ATP or GTP and the non-hydrolysable ATP analogue AMPPcP. Neither inactivating mutagenesis of archease nor substitution or omission of cofactors affected the obtained binding constants to a significant extent. Unchanged affinity for archease in the presence of non-hydrolysable AMPPcP indicates that ATP hydrolysis is not required for recruitment of archease to the ligase complex.

Extended Data Figure 6 Mechanistic aspects of the stimulation of RTCB by archease.

a, Native PAGE of Flag–RTCB–RNA adducts formed in the presence of wild-type (WT) or mutant (D39A, K144A, T147A) archease at 5 min (top). Denaturing PAGE of the same reactions (bottom). b, Denaturing PAGE of Flag–RTCB–RNA adducts isolated from the gel shown in a. (Note that part of Extended Data Fig. 6b is identical to Fig. 3b, in which the results for the two further mutants were not shown for clarity.) c, Antibody supershift assays (anti-Flag) retards Flag–RTCB, anti-His6 retards recombinant archease, anti-control (Ctrl)) of Flag–RTCB–RNA adducts assembled in the presence of mutant (D39A) archease (30 min). Incubation with wild-type archease demonstrates that antibody incubation does not inhibit ligation. Arrowheads indicate the position of supershifted complexes. A marked supershift in the presence of anti-Flag antibodies indicates that the visualized complexes contain Flag–RTCB.

Extended Data Figure 7 Mechanistic aspects of RTCB-catalysed RNA ligation and characterization of mutant versions of DDX1.

a, Tentative model illustrating the mode of action of human archease (indicated by the green shaded area) in the RTCB reaction cycle. Before activating RNA 2′,3′ cyclic phosphate termini for ligation, RTCB undergoes guanylation resulting in a guanylated form of the enzyme (i). GMP is then transferred to the 3′ end of the RNA substrate (ii). After ligation the active site of RTCB is occupied by the products of the reaction (iii). Another round of catalysis requires guanylation of the RTCB active site that depends on archease and is extensively stimulated by ATP hydrolysis by DDX1. b, Domain structure of DDX1 (DEAD helicase domain in dark red, SPRY domain in grey and carboxy-terminal helicase domain in blue). DDX1 was mutagenized in the ATPase A motif (K52N, predicted to interfere with ATP binding), in the ATPase B motif (E371Q, predicted to interfere with ATP hydrolysis), or in the C-terminal helicase domain (R557K and H601Q, predicted to interfere with helicase activity)21. c, SDS–PAGE of a 10 μl aliquot of Flag–DDX1 purified from HEK293 cells. d, ATPase assay of wild-type (WT) and mutant (K52N, E371Q) Flag–DDX1 (top) quantified signals are displayed in the bottom panel (mean ± s.e.m., N = 4). e, SDS–PAGE analysis of Flag–DDX1 (wild type, K52N and E371Q) crosslinked to [α-32P]ATP. The top panel (autoradiography) reveals affected ATP crosslinking for the two mutants K52N and E371Q whereas the bottom panel (Coomassie blue) confirms equal loading of the purified complexes. f, RNA filter binding assay of wild-type and mutant (K52N, E371Q) Flag–DDX1 (left). Quantified signals obtained for wild-type Flag–DDX1 in the presence of ATP were set to 100% (right; mean ± s.e.m., N = 9).

Extended Data Figure 8 Effect of mutagenesis of DDX1 on the activity of the human RNA ligase complex.

a, SDS–PAGE of wild-type (WT) or mutant (K52N, E271Q, R577K, H601Q) Flag–DDX1 incubated with archease and [α-32P]GTP. b, Ligase assays (100 nM substrate) of 20 nM wild-type or mutant (K52N, E271Q, R577K, H601Q) Flag–DDX1 in the presence of archease (10 µM) and 0.5 mM ATP (top) or AMPPnP (bottom). c, Michaelis–Menten kinetics of wild-type or mutant (K52N, E271Q, R577K, H601Q) DDX1 (20 nM RNA ligase complex, 10 µM archease). Reaction rates were estimated as indicated in Extended Data Fig. 9. d, Michaelis–Menten parameters (mean ± s.e.m.) were obtained by nonlinear regression using GraphPad. kcat (catalytic constant) values were obtained by multiplication of velocity of enzyme-catalysed reaction at infinite concentration of substrate (Vmax) values by the concentration of ligase complex. e, Michaelis–Menten parameters of wild-type and mutant Flag–DDX1 (mean ± s.e.m.). Comparisons between wild-type and mutant Flag–DDX1 were performed by z-test. The z-statistics were computed as (<wild type> − <mutant>)/(sqrt(s.e.m.(<wild type>)2 + s.e.m.(<mutant>)2). From these z values we obtained Bonferroni-corrected two-sided P values (not significant, NS; P > 0.05, ***P < 0.0004). The mutants K52N and E371Q exhibit a marked reduction of kcat, with only insignificant changes of the Michaelis constant (Km), indicating that ATP binding and hydrolysis contribute to catalytic steps (such as guanyl transfer) of human RNA ligase rather than substrate binding.

Extended Data Figure 9 Determination of RNA ligase reaction rates for the estimation of Michaelis–Menten parameters of Flag–DDX1 complexes.

The progress of reactions (20 nM RNA ligase complex, 100 nM to 4 µM substrate, 10 µM archease) was recorded for 15 min assuming near-linear behaviour of the reaction in this time frame (mean ± s.e.m., N = 2). Reaction rates were determined by linear regression, reaction rates (considering both concatemerized and circular product species) for each preparation (wild type (WT), K52N, E371Q, R577K, H601Q) are stated below each graph (nM s−1; mean ± s.e.m.).

Extended Data Figure 10 Depletion of archease does not indirectly impair tRNA processing by concomitant depletion of RTCB.

a, Quantitative polymerase chain reaction with reverse transcription (qRT–PCR) of RTCB and archease mRNAs in the cells used to prepare the extracts for the experiments shown in Fig. 4a. Expression levels are plotted as relative amounts of transcripts with respect to control-treated cells (mean ± s.d., N = 3 technical replicates). b, Western blot of RTCB in extracts used for the experiments shown in Fig. 4a. c, Western blot of tRNA ligase complex members in extracts used for the experiments shown in Fig. 4d. d, qRT–PCR of RTCB and archease mRNA in cells analysed in Fig. 4b. Expression levels are plotted as relative amounts of transcripts with respect to control-treated cells (mean ± s.d., N = 3 technical replicates).

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Popow, J., Jurkin, J., Schleiffer, A. et al. Analysis of orthologous groups reveals archease and DDX1 as tRNA splicing factors. Nature 511, 104–107 (2014). https://doi.org/10.1038/nature13284

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