Exoribonuclease Rat1 and Activating Partner Rai1

Ty J. Boyd'20, Peter E. Reinhart'20


I. Introduction

Introduction: 5’ to 3’ exoribonucleases are a large family of conserved enzymes in eukaryotes. These enzymes have important functions in RNA metabolism and interference. Schizosaccharomyces pombe Rat1, or XRN 2 in humans, also plays a major role in the termination of RNA Polymerase II. Rat1 is stimulated by its activating partner, Rai1. In vitro, Rat1 is unstable and loses nuclease function upon pre-incubation unless in complex with Rai1. The Rat1-Rai1 complex forms a more stable secondary structure, allowing for the efficient degradation of RNAs. Independent of its interaction with Rat1, Rai1 has pyrophosphohydrolase activity toward the 5’ triphosphorylated end of RNA. This type of activity is crucial for degradation of mRNA.

II. Rat1 and Rai1 Structure

Rat1 Active Site

S. pombe Rat1 has two conserved segments. These two sequences make up a large domain of the protein . Of these conserved sequences, the first segment makes up the majority of the enzyme’s active site. This region shares has several weak nuclease homologs in the PDB (i.e RNase H). Like these other nucleases, the active site is made up of a similar cluster of acidic residues. Suggesting that these proteins share the same catalytic activity. The second segment surrounds the active site in a "pocket-like fashion" not found in other homologs, making few direct contributions active site.

Xiang et al, Figure 1c

The unique pocket-like structure is responsible for Rat1’s inability to perform endonuclease activity. Thus, providing a Molecular explanation for the exclusivity of its exonuclease activity.

Tower Domain

Rat1 contains a notably large helix (αD) that extends 30Å out from the protein. This structure is known as the of Rat1. While the C-terminal of the tower extends far from the protein, the N-terminus contains several conserved residues to the active site. Interestingly, Xrn1 homologs have deletions in the C-terminus of the enzyme, suggesting that tower domain may serve functions that are specific to Xrn2 such as Pol II termination.

Rai1 Structure

Structures of Rai1 and its homolog DOM3Z contain two “highly twisted” β-sheets and several nearby helices. This motif, along with the unique sequences of these proteins would suggest a new protein fold. More importantly, this structure may suggest catalytic function. The residues of in addition to the main chain carbonyl of Leu240 and two water molecules form the coordination sphere of a cation.

Xiang et al, Figure 3c

The pocket here serves as an active site for Rai1 and its homolog. Interestingly, the conformation of the active site is almost identical in DOM3Z, despite the protein not interacting with Rat1. This suggests the active site functions independently of the binding of Rat1. Rat1-Rai1


Rai1 is bound 30Å away on the face opposite to Rat1’s active site. The main interactions involve the C-terminal segment of Rat1 and the β8-αE segment of Rai1 (as well as strand β4). Mutations in the residues of in Rai1 greatly reduced interactions between the Rat1 and Rai1 . Mutation of Rat1 residues on the interface had similar consequences.

III.Rai1 Pyrophosphohydrolase Activity

Data from exoribonuclease assays show that the catalytic activity of Rai1 like that of a 5’ RNA pyrophosphohydrolase. This activity turns a 5’-triphosphate group into a 5’-monophosphate group, thus allowing the RNA to become a substrate for Rat1. Interactions between GDP and the [DOM3Z active site], Rai1 analog, have helped to define the substrate binding mode of the active site of Rai1. The phosphate of GDP makes strong interactions with the dipole of helix αB near the N-terminus. Direct interactions are also made with the side chain of Arg94(DOM3Z Arg132). The β phosphate makes interactions with the same Arginine as well as Gln263(DOM3Z Gln280) in the αF helix. Interestingly, the hydroxyls as well as the guanine base are not recognized by any conserved residues, though the ribose does interact with Trp93 (DOM3αZ Trp131) via Van der Waals. Finally, conserved Gln199 (DOM3Z Glu234) is located near the phosphates of GDP and may serve a catalytic role in the of Rai1.

Xiang et al, Figure 3d,e

IV.Termination of RNAPII by Rat1

One of the most important roles of Rat1 is its function in the termination of RNA Polymerase II (RNAPII) during transcription. Loss of Rat1(or Rai1) has been shown to result in defective and inefficient transcription termination. The torpedo model of transcription termination suggests that Rat1 (XRN2) enters a polyA cleavage site and degrades the nascent RNA until eventually displacing RNAPII from DNA. More specifically, it has been suggested that the length of the nascent RNA degraded by Rat1 is correlated positively with the overall efficiency of RNAPII termination. This suggests that, not only does the exoribonuclease activity of Rat1 allow it to gain access to the polymerase, but it may also play a role in developing a “driving force” in order to displace the polymerase. Interestingly, Rat1’s inability to terminate the RNAP in E. coli suggests interactions between the polymerase and Rat1 must maintain a certain level of specificity. These interactions are not yet well understood due to a lack of structural studies of the Rat1-RNAPII complex.

V. References

1) Xiang, S., Cooper-Morgan, A., Jiao, X., Kiledjian, M., Manley, J. L., & Tong, L. (2009). Structure and function of the 5’?3’ exoribonuclease Rat1 and its activating partner Rai1. Nature, 458(7239), 784–788. http://doi.org/10.1038/nature07731;

2)Nagarajan, V. K., Jones, C. I., Newbury, S. F., & Green, P. J. (2013). XRN 5’?3’ exoribonucleases: Structure, mechanisms and functions. Biochimica et Biophysica Acta, 1829(0), 590–603. http://doi.org/10.1016/j.bbagrm.2013.03.005

3)Park, J., Kang, M., & Kim, M. (2015). Unraveling the mechanistic features of RNA polymerase II termination by the 5?-3? exoribonuclease Rat1. Nucleic Acids Research, 43(5), 2625–2637. http://doi.org/10.1093/nar/gkv133

4)Lemay, J.-F., & Bachand, F. (2015). Fail-safe transcription termination: Because one is never enough. RNA Biology, 12(9), 927–932. http://doi.org/10.1080/15476286.2015.1073433Lemay, J.-F., & Bachand, F. (2015). Fail-safe transcription termination: Because one is never enough. RNA Biology, 12(9), 927–932. http://doi.org/10.1080/15476286.2015.1073433

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