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

Abstract

The 5’→3’ exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference1–5. XRN2, or Rat1 in yeast6, functions primarily in the nucleus and also plays an important role in transcription termination by RNA polymerase II (Pol II)7–14. Rat1 exoribonuclease activity is stimulated by the protein Rai115, 16. Here we report the crystal structure at 2.2 Å resolution of S. pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homolog 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 more effectively degrade RNAs with stable secondary structure. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N-terminus) domain-containing nucleases17–20. 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 mRNA degradation in bacteria21, but ours is the first demonstration of this activity in eukaryotes and suggests that Rai1/DOM3Z may have additional important functions in RNA metabolism.

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

“…The mechanism underlying the inactivation of the exonuclease by this mutation at non-permissive temperature (above 34 C) is not well understood. As previously suggested from structural studies, 23 we found that this inactivation is in part due to the instability of the mutated protein. Indeed, as revealed by Western blot analyses shown in Fig.…”

“…29 Thus, its implication in this alternative nuclear mRNP QC is conceivable. In contrast, our data indicate clearly that Rai1p, the Rat1p cofactor specialized in removing unmethylated caps, 23,24 is not involved in the production of uncapped HXK1 mRNPs since no transcript recovery could be observed in the rai1D strain (Fig. 5A).…”

Transcription termination by nuclear RNA polymerases

“…In both cases, inactivation of the exonuclease resulted in defects in termination, as measured by ChIP and/or nuclear run-on experiments, both methods measuring RNAPII density throughout transcribed genes. Rat1 and its cofactor Rai1 (see Xiang et al 2009) are strongly associated with the 39-end of genes, although in a manner independent of Ser2 CTD phosphorylation since a deletion of CTK1 does not affect their association (Kim et al 2004b). Recruitment of Rat1 is more likely indirect, through 39-end processing factors.…”

Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae

“…Previous work has identified the Rai1 protein as an enzyme that can function to remove the cap structure from mRNA in S. cerevisiae (25,26). In contrast to the Dcp1/Dcp2 complex that preferentially functions on a cap with an N7 methyl moiety and release m 7 Gpp, Rai1 preferentially targets mRNAs with unmethylated caps and releases the entire cap structure GpppN, although it can function on a methylated cap to release m 7 GpppN to a lesser extent (26).…”