The human RNA kinase hClp1 is active on 3′ transfer RNA exons and short interfering RNAs


RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells1. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer2, synthetic siRNAs display a 5′ OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5′ end3,4,5. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing6 and messenger RNA 3′-end formation7 machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5′ end of the 3′ exon during human tRNA splicing8, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3′-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: hClp1 is an siRNA kinase.
Figure 2: Immunodepletion of hClp1 reduces target mRNA cleavage by non-phosphorylated siRNAs.
Figure 3: hClp1 phosphorylates the 5′ end of 3′ tRNA exons.
Figure 4: Silencing of hClp1 impairs mature tRNA formation.


  1. 1

    Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411, 494–498 (2001)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Nykänen, A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001)

    Article  Google Scholar 

  4. 4

    Pellino, J. L., Jaskiewicz, L., Filipowicz, W. & Sontheimer, E. J. ATP modulates siRNA interactions with an endogenous human Dicer complex. RNA 11, 1719–1724 (2005)

    CAS  Article  Google Scholar 

  5. 5

    Pham, J. W. & Sontheimer, E. J. Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway. J. Biol. Chem. 280, 39278–39283 (2005)

    CAS  Article  Google Scholar 

  6. 6

    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 

  7. 7

    de Vries, H. et al. Human pre-mRNA cleavage factor IIm contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J. 19, 5895–5904 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Zillmann, M., Gorovsky, M. A. & Phizicky, E. M. Conserved mechanism of tRNA splicing in eukaryotes. Mol. Cell. Biol. 11, 5410–5416 (1991)

    CAS  Article  Google Scholar 

  9. 9

    Tanabe, S. et al. AF10 is split by MLL and HEAB, a human homologue to a putative Caenorhabditis elegans ATP/GTP-binding protein in an invins(10;11)(p12;q23q12). Blood 88, 3535–3545 (1996)

    CAS  PubMed  Google Scholar 

  10. 10

    Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951 (1982)

    CAS  Article  Google Scholar 

  11. 11

    Wang, L. K. & Shuman, S. Domain structure and mutational analysis of T4 polynucleotide kinase. J. Biol. Chem. 276, 26868–26874 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Shuman, S. & Hurwitz, J. 5′-Hydroxyl polyribonucleotide kinase from HeLa cell nuclei. J. Biol. Chem. 254, 10396–10404 (1979)

    CAS  PubMed  Google Scholar 

  13. 13

    Leuschner, P. J., Ameres, S. L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006)

    CAS  Article  Google Scholar 

  14. 14

    Abelson, J., Trotta, C. R. & Li, H. tRNA splicing. J. Biol. Chem. 273, 12685–12688 (1998)

    CAS  Article  Google Scholar 

  15. 15

    Nishikura, K. & De Robertis, E. M. RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. J. Mol. Biol. 145, 405–420 (1981)

    CAS  Article  Google Scholar 

  16. 16

    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 

  17. 17

    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  PubMed  Google Scholar 

  18. 18

    Winicov, I. & Button, J. D. Nuclear ligation of RNA 5′-OH kinase products in tRNA. Mol. Cell. Biol. 2, 241–249 (1982)

    CAS  Article  Google Scholar 

  19. 19

    Zillman, M., Gorovsky, M. A. & Phizicky, E. M. HeLa cells contain a 2'-phosphate-specific phosphotransferase similar to a yeast enzyme implicated in tRNA splicing. J. Biol. Chem. 267, 10289–10294 (1992)

    Google Scholar 

  20. 20

    Tomari, Y. & Zamore, P. D. Perspective: machines for RNAi. Genes Dev. 19, 517–529 (2005)

    CAS  Article  Google Scholar 

  21. 21

    Wickens, M. & Gonzalez, T. N. Molecular biology. Knives, accomplices, and RNA. Science 306, 1299–1300 (2004)

    CAS  Article  Google Scholar 

  22. 22

    Minvielle-Sebastia, L., Preker, P. J., Wiederkehr, T., Strahm, Y. & Keller, W. The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3′-end formation. Proc. Natl Acad. Sci. USA 94, 7897–7902 (1997)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Aranda, A. & Proudfoot, N. Transcriptional termination factors for RNA polymerase II in yeast. Mol. Cell 7, 1003–1011 (2001)

    CAS  Article  Google Scholar 

  24. 24

    West, S., Gromak, N. & Proudfoot, N. J. Human 5′→3′ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 432, 522–525 (2004)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Kim, M. et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517–522 (2004)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Stevens, A. & Poole, T. L. 5′-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5′-exonuclease-1. J. Biol. Chem. 270, 16063–16069 (1995)

    CAS  Article  Google Scholar 

  27. 27

    Martinez, J., Patkaniowska, A., Urlaub, H., Lührmann, R. & Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Leuschner, P. J., Ameres, S. L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006)

    CAS  Article  Google Scholar 

Download references


We would like to thank all members of our laboratory, J. Penninger and E. Arn for their encouragement and suggestions during the completion of this work. We also thank K. Heindl for experimental help; K. Mechtler for mass spectrometry analysis; A. Schleiffer for bioinformatics analysis; W. Keller for his gift of the polyclonal antibody against hClp1; and P. Leuschner, S. Ameres, T. Tuschl, G. Superti-Furga, T. de Lange, W. Aufsatz, L. Ringrose and J. M. Peters for their comments on the manuscript. S.W. is a postdoctoral fellow funded by IMBA, the Institute of Molecular Biotechnology of the Austrian Academy of Sciences. J.M. is supported by IMBA.

Author Contributions S.W. purified and identified hClp1, evaluated and characterized the protein, and designed, performed and analysed all experiments regarding hClp1’s function in tRNA splicing. J.M. adapted and established the siRNA-kinase assay and performed and analysed the experiments demonstrating hClp1’s role in RISC assembly and RNA target cleavage. Both authors discussed the results and contributed equally in writing and revising the manuscript.

Author information



Corresponding author

Correspondence to Javier Martinez.

Ethics declarations

Competing interests

Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14 and Supplementary Table 1 with Legends, Supplementary Materials and Methods and additional references. (PDF 4284 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Weitzer, S., Martinez, J. The human RNA kinase hClp1 is active on 3′ transfer RNA exons and short interfering RNAs. Nature 447, 222–226 (2007).

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing