H. sapiens Exoribonuclease
of viral RNA
Blake Calcei '16, Brandon January '15 and Alexander McQuiston
'16
Contents:
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.
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