1. Immunology and Inflammation
  2. Structural Biology and Molecular Biophysics
Download icon

Unified mechanisms for self-RNA recognition by RIG-I Singleton-Merten syndrome variants

  1. Charlotte Lässig
  2. Katja Lammens
  3. Jacob Lucián Gorenflos López
  4. Sebastian Michalski
  5. Olga Fettscher
  6. Karl-Peter Hopfner  Is a corresponding author
  1. Ludwig-Maximilians-Universität München, Germany
  2. Gene Center, Ludwig-Maximilians-Universität München, Germany
  3. Center for Integrated Protein Science Munich, Germany
Research Advance
Cite this article as: eLife 2018;7:e38958 doi: 10.7554/eLife.38958
4 figures, 1 video, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
The RIG-I Singleton-Merten syndrome variant C268F signals in response to endogenous dsRNA.

(A) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + standard error of the mean (SEM). (B) Fluorescence anisotropy changes measured after titrating RIG-I or RIG-I C268F in the presence or absence of ATP into solutions containing a fluorescently labeled 14mer dsRNA. All binding curves were fit to a one-site binding equation using R. n = 4, error bars represent mean values ± standard deviation (SD).

https://doi.org/10.7554/eLife.38958.002
Figure 1—figure supplement 1
Location of RIG-I amino acid substitutions used in Figure 1.

A RIG-I-RNA-ADP·BeF3 structure (PDB: 5E3H) served as scaffold (Jiang et al., 2011). The RIG-I SF2 sub-domains (1A, 2A and 2B) are colored in light blue and dark blue. The RD is depicted in cyan and 2CARD is indicated in yellow. Mutated amino acid side chains are depicted in orange. C268 and K270 are located in the SF2 motif I (‘P-loop’) and mutation of K270 reduces ATP binding (Rawling et al., 2015). Mutation of motif II E373 slows down ATP hydrolysis but keeps the molecule's ATP-binding properties intact. T347 recognizes the RNA backbone and its mutation impedes RNA-dependent signaling of wild type RIG-I (Lässig et al., 2015). C268 and E373 are mutated in atypical Singleton-Merten syndrome.

https://doi.org/10.7554/eLife.38958.003
Figure 1—figure supplement 2
Comparison of the autoimmune signaling activity of RIG-I Singleton-Merten syndrome variants.

(A) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + SEM. Overall, our results corroborate the findings reported by Jang et al. (2015) and by Lässig et al. (2015) in a concentration-dependent manner. (B) Normalized IFN-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells that were co-transfected with the RIG-I SMS variant C268F, p-125luc/pGL4.74 reporter plasmids and increasing amounts of signaling-incompetent versions of RIG-I. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after second transfection. All ratios were normalized to the RIG-I C268F-only control. n = 4, error bars represent mean values + SEM.

https://doi.org/10.7554/eLife.38958.004
Figure 2 with 1 supplement
The RIG-I Singleton-Merten syndrome variant C268F is catalytically dead and has reduced ATP-binding-properties.

(A) ATP hydrolysis activity of RIG-I, the RIG-I Singleton-Merten syndrome (SMS) variant C268F and the RIG-I motif I and II mutants K270I and E373Q. RIG-I proteins were incubated with [γ-32P]-ATP in the presence or absence of a 12mer dsRNA for 15 min at room temperature and free phosphate was separated from ATP by thin layer chromatography. (B) Affinity of RIG-I, RIG-I C268F and the RIG-I motif I and II mutants to MANT-ATP or MANT-ATPγS measured by tryptophan fluorescence Förster resonance energy transfer to the MANT-nucleotide. Proteins were incubated with increasing amounts of nucleotides in the presence or absence of a 14mer dsRNA. MANT fluorescence was recorded minus a MANT-nucleotide-only control. n = 4, error bars represent mean values ± SD. (C) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + SEM.

https://doi.org/10.7554/eLife.38958.005
Figure 2—figure supplement 1
Location of RIG-I amino-acid substitutions used in Figure 2.

A RIG-I-RNA-ADP·BeF3 structure (PDB: 5E3H) served as scaffold (Jiang et al., 2011). The RIG-I SF2 sub-domains (1A, 2A and 2B) are colored in light blue and dark blue. The RD is depicted in cyan and 2CARD is indicated in yellow. Mutated amino acid side chains are depicted in orange. Q247 and R244 are located within the SF2 Q-motif and participate in ATP base recognition. C268 is mutated in atypical Singleton-Merten syndrome.

https://doi.org/10.7554/eLife.38958.006
Figure 3 with 1 supplement
The RIG-I Singleton-Merten syndrome variant C268F induces amino acid side chain rearrangements within the active site that interfere with nucleotide binding.

(A) ATP-binding pockets of the RIG-I Singleton-Merten syndrome (SMS) variant C268F (left and middle panels) and the RIG-I wild type (right panel) bound to a 14mer dsRNA. The RIG-I SF2 sub-domains are colored in light gray or light blue (1A and 2A) and dark blue (2B). The RD is depicted in cyan and 2CARD is indicated in yellow. (B) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + SEM.

https://doi.org/10.7554/eLife.38958.008
Figure 3—figure supplement 1
Structural comparison of the RIG-I Singleton-Merten syndrome variant C268F with wild type RIG-I.

(A) Alignment of RIG-I Δ2CARD C268F (light gray) with wild type RIG-I Δ2CARD (color code as in Figure 1—figure supplement 1) PDB 5E3H (Jiang et al., 2011). ADP·Bef3 and Mg2+ are co-crystalized with wild type RIG-I Δ2CARD but not with RIG-I Δ2CARD C268F. (B) 2Fo − Fc electron density of residues within the ATP-binding pocket of RIG-I Δ2CARD C268F at a contour level of 1σ.

https://doi.org/10.7554/eLife.38958.009
Model for the impact of Singleton-Merten syndrome mutations on self-RNA-induced RIG-I signaling.

(A) In healthy cells, wild type RIG-I occurs in a signal-off state in which 2CARD is shielded by binding to the insertion domain of SF2. Binding of RIG-I to self-RNAs is efficiently prevented through ATP-turnover-induced dissociation (for a detailed model on self- vs non-self RNA discrimination see also Lässig et al. (2015). (B) RIG-I Singleton-Merten syndrome (SMS) mutations either slow down ATP hydrolysis and stabilize the ATP-state (E373A, left side) or mimic the ATP-bound state (C268F, right side), and thus allow formation of the RIG-I signal-on state. In both cases, loss of ATP hydrolysis enhances the interaction with self-RNA and therefore results in pathogenic signaling. SMS mutations are indicated with a yellow or orange star.

https://doi.org/10.7554/eLife.38958.011

Videos

Video 1
Crystal structure of RIG-I Δ2CARD C268F and close-up of the active site.

The Singleton-Merten syndrome (SMS) mutation F268, as well as K270 and E702, are represented by a stick model. Theoretic locations of ADP·BeF3 and Mg2+ are indicated in faint sticks and spheres, respectively, according to a superposition with RIG-I Δ2CARD in complex with RNA and nucleotide analogue (PDB 5E3H). K270 is located at the Mg2+-binding site, whereas E702 occupies the BeF3 (ATP γ-phosphate) position.

https://doi.org/10.7554/eLife.38958.010

Tables

Table 1
Affinities of different RIG-I mutants to MANT-ATP or MANT-ATPγS in the presence or absence of a 14mer dsRNA. n.d., not determined, n.f., no fit possible as no saturation was reached.
https://doi.org/10.7554/eLife.38958.007
ProteinMANT-ATPMANT-ATPγS
RIG-I72 ± 13 µM58 ± 7 µM
RIG-I + RNAn.d.11 ± 1 µM
RIG-I E373Q72 ± 13 µMn.d
RIG-I E373Q + RNA28 ± 5 µMn.d
RIG-I K270I298 ± 81 µMn.d
RIG-I K270I + RNAn.f.n.d
RIG-I C268F166 ± 34 µM116 ± 13 µM
RIG-I C268F + RNAn.f.147 ± 55 µM
Key resources table
Reagent type (species)
or source
DesignationSource or referenceIdentifiersAdditional information
Cell line (human)HEK293T RIG-I KOZhu et al. (2014)Growth in Dulbecco's Modified Eagle
Medium (DMEM) supplemented with
10% fetal bovine serum (FBS) as
monolayer
Strain, strain
background
(Escherichia coli)
BL21 (DE3) RosettaNovagene
Strain, strain
background
(Escherichia coli)
DH10multiBacGenevaBiotech
Strain, strain
background
(Spodoptera frugipeda)
Sf21 insect cellsThermo Fisher
Scientific
11497013Growth in SF-900 III
serum-free medium
Strain, strain
background
(Trichoplusia ni)
High Five insect cellsThermo Fisher
Scientific
B85502Growth in Express Five serum-free
medium supplemented with
10 mM L-glutamine
Recombinant
DNA reagent
pcDNA5/FRT/TOThermo Fisher
Scientific
V652020
Recombinant
DNA reagent
pcDNA5/FRT/TO-FLAG/
HA-RIG-I and various
mutants of the same
construct
Lässig et al. (2015)
and this paper
Progenitors: PCR, DDX58
(cDNA) and pcDNA5/FRT/TO
Recombinant
DNA reagent
p-125lucYoneyama et al. (1996)Firefly luciferase controlled
by an interferon-β promoter
Recombinant
DNA reagent
pGL4.74PromegaE6921Constitutive expression of a
Renilla luciferase
Recombinant
DNA reagent
pFBDMBerger et al. (2004)
Recombinant
DNA reagent
pFBDM-His-RIG-I
and various mutants of
the same construct
Lässig et al. (2015)
and this paper
Progenitors: PCR, DDX58 (cDNA)
and pFBDM
Recombinant
DNA reagent
pETM11-SUMO3GFPEMBL Heidelberg,
H. Besir
https://www.embl.de/pepcore/pepcore_services/cloning/sumo/
Recombinant
DNA reagent
pETM11-SUMO3-RIG-I-
Δ2CARD-C268F
This paperProgenitors: PCR, DDX58
(cDNA) and pETM11-SUMO3GFP
Sequence-based
reagent
19mer 5' triphosphate
dsRNA
InvivoGentlrl-3prna1 µg/mL, 5’-pppGCAUGC
GACCUCUGUUUGA-3
Sequence-based
reagent
14mer dsRNADharmacon5'-CGACGCUAGCGUCG-3'
Sequence-based
reagent
Cy3-hpRNABiomers5'-Cy3-CCACCCGCCCCCCUAGU
GAGGGGGGCGGGCC-3'
Chemical
compound, drug
Lipofectamine 2000Thermo Fisher
Scientific
11668019Used at 2.5x excess compared
to RNA/DNA mass
Chemical
compound, drug
MANT-ATPJena BioscienceNU-202
Chemical
compound, drug
MANT-ATPγSJena BioscienceNU-232
Chemical
compound, drug
[γ-32P]ATPHartmann AnalyticSRP-30110 nM spiked with
3 mM unlabeled ATP
Commercial
assay or kit
Dual-Luciferase
Reporter Assay
System
PromegaE1910
Software,
algorithm
XDS, XSCALEKabsch (2010)http://xds.mpimf-heidelberg.mpg.de/
Software,
algorithm
PHASERMcCoy et al. (2007);
Winn et al. (2011)
http://www.ccp4.ac.uk/
Software,
algorithm
CootEmsley et al. (2010)https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software,
algorithm
PHENIXAfonine et al. (2012)https://www.phenix-online.org/
Software,
algorithm
PymolSchrödingerhttps://pymol.org/2/
Software,
algorithm
RR Development Core Team (2013)https://www.r-project.org/
Table 2
Data collection and refinement statistics.

Values in parentheses are for the highest resolution shell.

https://doi.org/10.7554/eLife.38958.012
Crystal 1Crystal 2
PDB code6GPG
Data collection
Space groupP212121P6522
Wavelength (Å)1.001.00
Cell dimensions
a, b, c (Å)112.1, 177.1, 314.8175.6, 175.6, 109.5
α, β, γ (°)90, 90, 9090, 90, 120
Resolution range (Å)47.2–3.3 (3.42–3.30)46.4–2.9 (3.00–2.89)
Rmerge (%)14.3 (112)7.6 (206)
II8.45 (1.28)19.72 (1.15)
CC1/299.8 (67.6)99.9 (99.7)
Completeness (%)95.3 (79.7)99.7 (97.4)
Redundancy3.38 (2.91)13.09 (13.21)
Refinement
Resolution (Å)3.32.9
No. reflections90,12122,649
Rwork/ Rfree22.8/28.321.4/25.9
No. atoms
 Macromolecules35,7305,810
 Ions102
Ramachandran statistics
 Favoured (%)92.7892.71
 Allowed (%)6.396.98
 Outliers (%)0.830.31
R.M.S deviations
 Bond lengths (Å)0.0110.009
 Angles (°)1.481.43
B-factors
 Macromolecules109.98139.89
 Ions105.23121.74

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)