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Acknowledgments: Funding: NSF-0330845, 0949361 to
M.A.C.; NSF 0949027 to R. W.; NSF ROA (1145355), University
of Iowa to A.A.F. Data are available in supplementary
materials and archived at National Center for Biotechnology
Information, NIH, GenBank (accession numbers: KF473465
to KF475237). The Instituto Nacional de Recursos Naturales-
Intendencia Forestal y de Fauna Silvestre del Perú authorized
collections (autorización no. 110-2008-INRENA-IFFS-DCB) and
exportation of specimens (permiso no. 011832-AG-INRENA).
T. Litwak assisted with illustrations. The authors declare no
conflicts of interest. USDA is an equal opportunity provider
Materials and Methods
Figs. S1 to S9
Tables S1 to S10
21 August 2013; accepted 29 January 2014
Structure of Human RNase L Reveals
the Basis for Regulated RNA Decay in
the IFN Response
Yuchen Han,* Jesse Donovan,* Sneha Rath,* Gena Whitney, Alisha Chitrakar, Alexei Korennykh†
One of the hallmark mechanisms activated by type I interferons (IFNs) in human tissues involves
cleavage of intracellular RNA by the kinase homology endoribonuclease RNase L. We report
2.8 and 2.1 angstrom crystal structures of human RNase L in complexes with synthetic and natural
ligands and a fragment of an RNA substrate. RNase L forms a crossed homodimer stabilized by
ankyrin (ANK) and kinase homology (KH) domains, which positions two kinase extension
nuclease (KEN) domains for asymmetric RNA recognition. One KEN protomer recognizes an identity
nucleotide (U), whereas the other protomer cleaves RNA between nucleotides +1 and +2.
The coordinated action of the ANK, KH, and KEN domains thereby provides regulated,
sequence-specific cleavage of viral and host RNA targets by RNase L.
Cells of higher vertebrates respond to path- ogens and damage by releasing interferons (IFNs), which activate protective programs
in surrounding cells. One of the ubiquitous protective programs in mammalian tissues involves
cleavage of intracellular RNA by a protein kinase
family receptor, RNase L (1). RNase L is a latent
endoribonuclease encoded by the hereditary prostate cancer 1 (HPC1) locus and activated by the second messenger, 2-5A (2′,5′-linked oligoadenylates
of variable length) (1). In human cells, 2-5As are
synthesized by IFN-induced 2-5A synthetases,
which serve as sensors of pathogen- and damage-associated double-stranded RNA (dsRNA) (2, 3).
The activation of RNase L thus depends on the
action of IFNs and accumulation of dsRNA.
Here, we report two crystal structures of
human RNase L (table S1). These structures
and complementary functional studies reveal
the mechanism of 2-5A sensing and RNA cleavage by RNase L and suggest that a similar mechanism of RNA cleavage operates during regulated
Ire1-dependent decay (RIDD) (4). We obtained
diffracting crystals of nearly full-length human
RNase L using cocrystallization with 2-5A, nucleotides, and an RNA 18-nucleotide oligomer
Cocrystallization with RNA18 was enabled by
the use of a catalytically inactive RNase L mutant
H672N. The final construct includes residues 21
to 719. A version of this construct with a wild-type
(WT) active site is catalytically active in solution.
We determined structures of two RNase L
complexes, which crystallized in different space
groups (table S1). Both complexes reveal the
same crossed homodimer that buries >8000 Å2 of
surface area (Fig. 1A). Previous solution studies
indicated that RNase L can form dimers and
higher-order oligomers (5). Modeling based on the
oligomer of Ire1 (6) predicts that the homodimers
of RNase L could form a similar assembly. The
kinase homology (KH) domain of RNase L has a
typical protein kinase fold with two globular
lobes (Fig. 1B). Adenosine diphosphate (ADP)
and b,g-methyleneadenosine triphosphate are
bound to the KH domain in the same conformation as ADP in the catalytically active protein
kinase Ire1 (fig. S1, A to C). Nonhydrolyzable
nucleotides and ATP exhibit the same effect on
RNase L (fig. S1D), indicating that ATP hydrolysis is not involved in RNase L regulation, as
suggested previously (7).
The KH domain lacks the conserved DFG
motif found in most protein kinases and con-
tains the DFD sequence, resembling protein ki-
nases Mnk1/2 (8). RNase L does not carry out
autophosphorylation (7), but it remains unknown
whether RNase L phosphorylates nonself targets.
To examine this possibility, we assayed phospho-
rylation of a nonspecific substrate, myelin basic
protein (MBP), using Ire1 as a control kinase.
Ire1 phosphorylated MBP, whereas RNase L was
inactive (fig. S1E), supporting the current consen-
sus that RNase L is a pseudokinase. The activa-
tion loop of RNase L contains only 13 amino acid
residues and is among the shortest in the human
protein kinome (fig. S3). The interlobe hinge and
the ATP pocket contact a unique helix from the
ankyrin (ANK)/KH linker (Fig. 1B and fig. S2).
These attributes of the KH domain likely reflect
adaptation to autophosphorylation-independent
control as a homodimerization scaffold.
Previous structural studies identified two different 2-5A binding sites in the ANK domain (5, 9).
The crystal structure of the entire RNase L now
reveals an unanticipated third site in the N lobe of
the KH domain (Fig. 1A and fig. S4, A to C). The
ANK and KH domains create a composite pocket for 2-5A binding, which exposes the 2′-end to
solvent to accommodate long 2-5A molecules
and anchors the 5′-end in the ANK domain (fig.
S5A). Although RNase L can recognize 5′-p and
5′-ppp groups, at saturating concentrations 2-5pA3
activates RNase L stronger than 2-5pppA3 (fig.
S5, B and C). The ANK/ANK homodimer binds
2-5A in a configuration that displays the phosphate p1 for recognition by the KH domain (fig.
S6). Mutagenesis of the KH/2-5A interface confirms that the N lobe is functionally involved
in 2-5A sensing (fig. S4D). The ANK domain contains a characteristic helix aI, which docks to
the KH domain in trans upon homodimerization (fig. S7). The aI/N-lobe interaction and an
ANK/N-lobe contact mediated by the residue
R238 also facilitate RNase L activation by 2-5A
(fig. S4, C and D).
The KH/KH and kinase extension nuclease
(KEN)/KEN interfaces resemble those in Ire1
(10). Mutagenesis confirmed that both interfaces
are important for 2-5A–dependent RNase L activation (Fig. 2A) and dimerization (fig. S8). The
KEN residues involved in catalysis in Ire1 (10, 11)
are structurally invariant in RNase L (Fig. 2B),
indicating that these enzymes share the same catalytic mechanism. However, the KEN domains
contain different a-helix/loop elements (HLE) (6),
implicated in RNA specificity (10). To examine
the HLE function, we shortened this element in
RNase L (DHLE). The DHLE mutant still cleaved
RNA but exhibited a decreased rate and a greater
preference for single-stranded RNA (ssRNA)
Department of Molecular Biology, Princeton University, 216
Schultz Laboratory, Princeton, NJ 08540, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: firstname.lastname@example.org