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Structure and function of the 5′→3′ exoribonuclease Rat1 and its activating partner Rai1

Nature volume 458, pages 784–788 (2009)Cite this article

Abstract

The 5′→3′ exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference1,2,3,4,5. XRN2, or Rat1 in yeast6, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7–14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 Å resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 Å resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases17,18,19,20. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5′ triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria21, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.

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Figure 1: Structure of the Rat1–Rai1 complex.
Figure 2: Biochemical and functional characterization of the Rat1–Rai1 interaction.
Figure 3: Structure of Rai1.
Figure 4: Biochemical evidence for pyrophosphohydrolase activity of Rai1.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates have been deposited at the Protein Data Bank with accession numbers 3FQD, 3FQG, 3FQI and 3FQJ.

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Acknowledgements

We thank R. Abramowitz and J. Schwanof for setting up the X4A and X4C beamlines and H. Robinson for setting up the X29A beamline at the NSLS, and S. Jia for providing the S. pombe genome. This research is supported in part by grants from the NIH to L.T. (GM077175), J.L.M. (GM28983) and M.K. (GM67005).

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York 10027, USA,

    Song Xiang, Amalene Cooper-Morgan, James L. Manley & Liang Tong

  2. Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854, USA,

    Xinfu Jiao & Megerditch Kiledjian

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Correspondence to Liang Tong.

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Xiang, S., Cooper-Morgan, A., Jiao, X. et al. Structure and function of the 5′→3′ exoribonuclease Rat1 and its activating partner Rai1. Nature 458, 784–788 (2009). https://doi.org/10.1038/nature07731

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