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

Abstract

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.

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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.

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Acknowledgements

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.

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Correspondence to Javier Martinez.

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

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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). https://doi.org/10.1038/nature05777

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