H. sapiens Exoribonuclease of viral RNA

Blake Calcei '16, Brandon January '15 and Alexander McQuiston '16


I. Introduction

The Homo sapiens exoribonuclease Xrn1 has important functions in transcription, RNA metabolism and RNA interference. Xrn1 is primarily cytosolic and functions in the degradation of decapped mRNAs. Xrn1 degrades RNA in a 5' to 3' direction. Xrn1 is part of the XRN family which is a highly conserved enzyme family found in eukaryotes.
Xrn1 is also used in the degradation of flavivirus genomic RNA (gRNA), with viruses including Dengue, West Nile, Yellow Fever, Japanese Encephalitis, and others. The Xrn1 enzymes degrade the viral gRNA in the 5' to 3' direction but halts at defined locations on the gRNAs 3' untranslated region which forms small, cut up sections of gRNA called subgenomic flaviviral RNA (sfRNA). The sfRNA produced is essential for the pathogenicity of the virus. The regions where the Xrn1 enzyme halts is due to pseudoknots created by the flavivirus RNA. The specific reason for Xrn1's inability to pass these pseudoknots and its specific mechanism is unknown, but it is believed that the structure of these knots is too complex for the Xrn1 enzyme to pass.  

II. General Structure

Xrn1 contains 6 domains including two linker domains. These domains include: CR1 , CR2 , D1 , D2 , D3 and D4 , respectively with linker domain between CR1 and CR2 . These domains interact to form a rectangular box structure with the linker domain being outside of the rectangle. CR1 is located in the center of the structure with CR2 flanking it forming a side of the box and defining the active site. The D1 through D4 domains flank the other side of CR1and are located at the C-terminus . The CR1-CR2 linker region is poorly conserved and is connected to CR1 through a 13 turn helix causing the linker to be far from the main structure.

III. RNA Recognition

The base of the first nucleotide of the ssRNA is stacked onto , and the 5' end of the ssRNA packs onto CR1. Within the CR1 there is a highly basic pocket that is lined with the conserved residues Lys93, Gln97, Arg100 and Arg101 . The target RNA must first be decapped and then marked on the 5' end by a monophosphate. The 5' phosphate group of the ssRNA inserts into the highly basic pocket of Xrn1. The Arg100 and Arg101 form hydrogen bonding interactions with the 5' phosphate oxygens . Then the CR1 domain closes allowing the catalytic process to begin. The two most important factors for Xrn1 recognition of ssRNA is the 5'-terminal stacking  with His41 and the monophosphate recognition by the CR1 highly basic binding pocket.

IV. Active Site and Domain Interaction

The active site of Xrn1 is located within the CR1, but is far away from domains D1-D4. The active site allows for the binding of an ion, which further supports the Xrn1 catalytic activity. The Mg2+ ion interacts with three acidic residues within the CR1; Glu177, Asp205, and Asp288 . Although the active site is far away, D1 may still affect active site function. Part of the D1's three-stranded B-sheets interacts with the N-terminus (part of the CR1), which is located near the active site . The N-terminus provides the active site with positive charges and hydrophilic residues. Therefore, the inability of D1 to interact with the N-terminus could interrupt its interaction with the active site. Also, D2-D4 may help maintain D1 conformation which could indirectly disrupt active site function.

V. References

Jeong Ho Chang, Song Xiang, Kehui Xiang, James L. Manley, and Liang Tong. 2011. Structural and Biochemical Studies of the 5' to 3' Exoribonuclease Xrn1. Natural Structure Molecular Biology. 18(3):270-276.

Martin Jinek, Scott M. Coyle, and Jennifer A. Doudna. 2011. Coupled 5' Nucleotide Recognition and Processivity in Xrn1-Mediated mRNA Decay. Molecular Cell. 41:600-608.

Erich G. Chapman, David A. Costantino, Jennifer L. Rabe, Stephanie L. Moon, Jeffrey Wilusz, Jay C. Nix, and Jeffrey S. Kieft. 2014. The Structural Basis of Pathogenic Subgenomic Falvivirus RNA (sfRNA) Production. Science. 344(6181):307-310.

Jeong Ho Chang, Song Xiang, and Liang Tong. 2011. 5'-3' Exonuclease Activity of XRNs. Ribonucleases, Nucleic Acids and Molecular Biology. Chapter 7, section 3:170.

Back to Top