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Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

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Abstract

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5′ and 3′ splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively1,2,3. A recent structure of an archaeal endonuclease complexed with a bulge–helix–bulge RNA has led to the unexpected hypothesis that catalysis requires a critical ‘cation-π sandwich’ composed of two arginine residues that serve to position the RNA substrate within the active site4. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5′ splice site requires the conserved cation-π sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.

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Figure 1: Hypothetical model of the cation-π sandwich of the eukaryotic tRNA-splicing endonuclease.
Figure 2: Cleavage of yeast pre-tRNA Phe and pre-tRNA Tyr by mutant tRNA-splicing endonuclease.
Figure 3: Model of the archaeal and eukaryotic tRNA-splicing endonuclease.

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Acknowledgements

We thank C. Romfo, A. Jacobson, J. Colacino and D. Glodowski for discussions and critical reading of the manuscript. Author contributions C.R.T. and S.V.P. contributed equally to this work and together with M.P. performed the mutagenesis, purification and testing of endonuclease complexes. C.R.T., H.L., S.V.P and S.W.P. wrote the manuscript together.

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  1. PTC Therapeutics, 100 Corporate Court, New Jersey, 07080, South Plainfield, USA

    Christopher R. Trotta, Sergey V. Paushkin, Meenal Patel & Stuart W. Peltz

  2. Department of Chemistry and Biochemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, 32306, USA

    Hong Li

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  1. Christopher R. Trotta
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  2. Sergey V. Paushkin
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  4. Hong Li
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Correspondence to Christopher R. Trotta.

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Trotta, C., Paushkin, S., Patel, M. et al. Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site. Nature 441, 375–377 (2006). https://doi.org/10.1038/nature04741

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