Article

HEXIM1 and NEAT1 Long Non-coding RNA Form a

Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response
Graphical Abstract
Authors
Mehdi Morchikh, Alexandra Cribier,
Raoul Raffel, ..., Olivier Schwartz,
Yamina Bennasser, Monsef Benkirane

Correspondence
mehdi.morchikh@igh.cnrs.fr (M.M.),
alexandra.cribier@igh.cnrs.fr (A.C.),
monsef.benkirane@igh.cnrs.fr (M.B.)

In Brief
Morchikh et al. identified a 7SK snRNP-
independent HEXIM1 complex
composed of NEAT1 RNA, DNA-PK, and
subunits of the nuclear paraspeckles,
which they named HDP-RNP. They found
that this complex is a key regulator of
DNA-mediated induction of the cGAS-
STING-IRF3 innate immune pathway.

Highlights
d HEXIM1, NEAT1, DNA-PK, and paraspeckle factors assemble
into an RNP complex (HDP-RNP)

d HDP-RNP regulates DNA-dependent activation of the cGAS-

STING-IRF3 pathway

d The HDP-RNP is involved in the KSHV-mediated innate

immune response

d ORF52 targets the HDP-RNP to prevent innate immune

response against KSHV

Morchikh et al., 2017, Molecular Cell 67, 113

August 3, 2017 2017 Elsevier Inc.
http://dx.doi.org/10.1016/j.molcel.2017.06.020
 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Molecular Cell

Article

HEXIM1 and NEAT1 Long Non-coding RNA Form

a Multi-subunit Complex that Regulates
DNA-Mediated Innate Immune Response
Mehdi Morchikh,1,6,* Alexandra Cribier,1,6,* Raoul Raffel,1 Sonia Amraoui,3 Julien Cau,4,5 Dany Severac,2 Emeric Dubois,2
Olivier Schwartz,3 Yamina Bennasser,1 and Monsef Benkirane1,7,*
1Institut de Genetique Humaine, Laboratoire de Virologie Moleculaire, Universite de Montpellier, CNRS UMR9002, 34000 Montpellier, France
2MGX-Montpellier GenomiX, 34094 Montpellier, France
3Institut Pasteur, Virus and Immunity Unit, URA CNRS 3015, 75015 Paris, France
4MRI-Montpellier, Biocampus, Montpellier, 34095 France
5IGH CNRS- Universite de Montpellier, UMR9002, 34396 Montpellier, France
6These authors contributed equally
7Lead Contact

*Correspondence: mehdi.morchikh@igh.cnrs.fr (M.M.), alexandra.cribier@igh.cnrs.fr (A.C.), monsef.benkirane@igh.cnrs.fr (M.B.)

http://dx.doi.org/10.1016/j.molcel.2017.06.020

SUMMARY rus recognition mainly relies on the detection of their nucleic

The DNA-mediated innate immune response under-
pins anti-microbial defenses and certain autoim-
mune diseases. Here we used immunoprecipitation,
mass spectrometry, and RNA sequencing to identify
a ribonuclear complex built around HEXIM1 and the
long non-coding RNA NEAT1 that we dubbed the
HEXIM1-DNA-PK-paraspeckle components-ribonu-
cleoprotein complex (HDP-RNP). The HDP-RNP con-
tains DNA-PK subunits (DNAPKc, Ku70, and Ku80)
and paraspeckle proteins (SFPQ, NONO, PSPC1,
RBM14, and MATRIN3). We show that binding of
HEXIM1 to NEAT1 is required for its assembly. We
further demonstrate that the HDP-RNP is required
for the innate immune response to foreign DNA,
through the cGAS-STING-IRF3 pathway. The HDP-
RNP interacts with cGAS and its partner PQBP1,
and their interaction is remodeled by foreign DNA.
Remodeling leads to the release of paraspeckle pro-
teins, recruitment of STING, and activation of
DNAPKc and IRF3. Our study establishes the HDP-
RNP as a key nuclear regulator of DNA-mediated
activation of innate immune response through the
cGAS-STING pathway.
INTRODUCTION
Innate immunity is the first line of defense against invading mi-
crobes. Upon recognition of the invader, host cells produce cy-
tokines, including type 1 interferons (IFNs), which culminates in
the synthesis of many antimicrobial proteins and chemokines.
This ensures the recruitment of immune cells to the site of infec-
tion and initiates adaptive immune responses (Stetson and
Medzhitov, 2006). The innate immune response triggered by vi-
acids. Cells possess compartmentalized proteins specialized
in the recognition of foreign nucleic acids. These nucleic acid
sensors belong to the pattern recognition receptor (PRR)
family that recognizes pathogen-associated molecular patterns
(PAMPs) and, subsequently, triggers a signaling cascade leading
to the production of pro-inflammatory cytokines to enable elim-
ination of the virus (Barbalat et al., 2011). Specific PRRs are
engaged depending on the nature of the viral genome and the
nucleic acid intermediates generated during their replica-
tion cycle.
There are many DNA sensors that have been identified to date,
including DNA-dependent activator of IFN-regulatory factors
(DAI), DDX41, gamma-interferon-inducible protein 16 (IFI16),
DNA-PK, DHX9, DHX36, DDX60, MRE11, AIM2, and cyclic-
GMP-AMP (cGAMP) synthase (cGAS) (Paludan, 2015). cGAS is
the principal sensor of cytosolic DNA (Gao et al., 2013; Sun
et al., 2013). Binding of DNA to cGAS and its DNA-binding part-
ner PQBP1 activates its enzymatic activity (Yoh et al., 2015).
cGAS catalyzes the production of cyclic guanosine monophos-
phate-AMP (cGAMP) from ATP and GTP. cGAMP is a second
messenger that binds to and activates the stimulator of inter-
feron genes (STING) in infected cells, as well as bystander cells
(Ablasser et al., 2013; Burdette and Vance, 2013; Burdette et al.,
2011; Gentili et al., 2015). STING dimer then recruits the TANK-
binding kinase 1 (TBK1) to phosphorylate and activate interferon
regulatory factor 3 (IRF3), ultimately leading to the expression
of type I IFNs (Burdette and Vance, 2013; Ishikawa and
Barber, 2008; Tanaka and Chen, 2012). DNA viruses, such as
herpes and vaccinia viruses, adenovirus, as well as retroviruses
including HIV-1, trigger innate immune response upon cGAS
recognition (Dai et al., 2014; Gao et al., 2013; Lam et al., 2014;
Wu et al., 2015). Because activation of cGAS elicits a potent anti-
viral response (Ma and Damania, 2016), viruses have evolved
countermeasure mechanisms to interfere with the cGAS-
cGAMP-signaling pathway and to establish successful infection.
Viruses are not the only source of PRRs inducing nucleic acids.
Indeed, host damage-associated molecular patterns (DAMPs)

Molecular Cell 67, 113, August 3, 2017 2017 Elsevier Inc. 1

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020
are nucleic acids, released in the context of cell or tissue dam-
age. DAMPs are sensed by PRRs, leading to the production of
inflammatory cytokines. These pathways have been shown to
play a role in many inflammatory diseases, including cancer
(Hernandez et al., 2016; Qian et al., 2014). Thus, understanding
the regulation of nucleic acid sensors is important not only as
efficient antiviral proteins but also as key players in inflammatory
diseases.
Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) is
an RNA-binding protein best known as the inhibitor of the posi-
tive transcription elongation factor b (P-TEFb) (Diribarne and
Bensaude, 2009), which controls transcription elongation by
RNA polymerase II. HEXIM1 achieves this function through bind-
ing to the 7SK RNA and sequestration of CyclinT1/CDK9 within
the 7SK small nuclear ribonucleoprotein particle (snRNP) com-
plex (Michels et al., 2003; Yik et al., 2003). Biochemical and func-
tional analyses have shown that HEXIM1 displays other cellular
functions in a P-TEFb-independent manner (Lew et al., 2012;
Shimizu et al., 2005). Here we identified a 7SK ribonucleoprotein
(RNP)-independent ribonuclear complex in which HEXIM1 binds
to the long non-coding RNA NEAT1 and constitutes a platform
for the recruitment of the DNA-PK complex (DNAPKc, Ku70,
and Ku80) and nuclear paraspeckle proteins (HDP-RNP). We
investigated here the function of the HDP-RNP, and we found
it plays an important role in regulating DNA-mediated induction
of the innate immune response through the cGAS-STING-IRF3
pathway.
RESULTS
HEXIM1 Nuclear Interactome Reveals an RNP Complex
Containing DNA-PK and Paraspeckle Components:
HDP-RNP
HEXIM1 is a transcription inhibitor and major partner and regu-
lator of the 7SK snRNP/P-TEFb complex (Michels et al., 2004;
Yik et al., 2003). However, only a small fraction of HEXIM1 is
engaged in this complex, suggesting that this protein may play
other cellular functions (Li et al., 2007). To investigate HEXIM1s
nuclear function, we purified HEXIM1 and its associated nuclear
partners. We cloned full-length HEXIM1 fused to FLAG and HA
epitope tags at its N terminus (and termed this construct
eHEXIM1). HeLa S3 cells expressing eHEXIM1 were used to pre-
pare nuclear extracts (NEs). Next, we subjected the NEs to
tandem immunoaffinity chromatography with anti-FLAG and
anti-HA antibodies. eHEXIM1-associated partners were visual-
ized by silver staining on SDS-PAGE (Figure 1A) and identified
by tandem mass spectrometry (Figure 1B). As expected, sub-
units of the 7SK snRNP/P-TEFb complex were recovered,
including CyclinT1, CyclinT2, CDK9, LARP7, and MEPCE (Fig-
ures 1A and 1B).
Interestingly, subunits of the DNA-PK complex (DNAPKc,
Ku70, and Ku80) (Davis et al., 2014) and proteins known to func-
tion in RNA biology and to assemble in subnuclear structures
called paraspeckles (Fox and Lamond, 2010; Nakagawa and
Hirose, 2012) were also recovered, including SFPQ, NONO,
PSPC1, RBM14, and MATRIN3 (Figures 1A and 1B). The interac-
tions between eHEXIM1 and the above-mentioned partners
were confirmed by probing the FLAG-immunoprecipitated
2 Molecular Cell 67, 113, August 3, 2017
(FLAG-IP) eHEXIM1 using specific antibodies (Figure 1C).
FLAG-IP using NEs prepared from HeLa S3 cells (Figure 1C,
lanes 1 and 4) and western blot using anti-RIF1 were used as
negative controls. Since HEXIM1 is an RNA-binding protein,
we sought to determine whether the identified interactions are
RNA dependent. NEs were thus treated with RNase prior to
eHEXIM1 IP. As expected, RNase treatment resulted in loss of
the P-TEFb subunits CyclinT1 and CDK9 (Figure 1C, compare
lane 5 to lane 6). Interestingly, DNA-PK complex subunits and
paraspeckle proteins were not recovered after RNase treatment;
therefore, their interaction with HEXIM1 is also RNA dependent
(Figure 1C, compare lane 5 to lane 6).
Since 7SK RNA is a major HEXIM1 partner, we questioned its
contribution to HEXIM1 interaction with the identified partners.
For this purpose, we analyzed the consequence of 7SK RNA
knockdown on HEXIM1 interaction with DNA-PK and para-
speckle components (Figure S1A). The presence of CDK9,
Ku70, DNAPKc, and SFPQ in NEs (lanes 15) and in FLAG-IPs
(lanes 610) was assessed by immunoblotting. The presence
of 7SK RNA in NEs (lower left panel) and in FLAG-IPs was quan-
tified by RT-PCR (qRT-PCR) (lower right panel). Consistently, all
the interactions were lost when eHEXIM1 was immunoprecipi-
tated from RNase-treated NEs (Figure S1A, compare lane 7 to
lane 8 and lower right panel). Knockdown of 7SK RNA resulted
in loss of CDK9 in the eHEXIM1 IP, but it had no effect on the
DNA-PK proteins Ku70 and DNAPKc or on the paraspeckle pro-
teins SFPQ and NONO (compare lane 9 to lane 10 and lower right
panel). RNase treatment and 7SK small interfering RNA (siRNA)
transfection resulted in reduced 7SK RNA levels in NEs (lower
left panel). Our experiments reveal that DNA-PK complex sub-
units and the paraspeckle proteins SFPQ, PSPC1, and NONO
are bona fide HEXIM1-interacting partners and that these inter-
actions are RNA dependent but do not depend on the previously
known HEXIM1-interacting RNA 7SK.
The identified interactions and their RNA dependency were
confirmed using endogenous proteins. Figure S1B shows that
IP of endogenous Ku70, similarly to HEXIM1, recovered not
only HEXIM1 and DNAPKc but also SFPQ and PSPC1 (compare
lane 3 to lane 5). These interactions were lost when Ku70
immunoprecipitate was RNase-treated, with the exception of
the interaction with DNAPKc (compare lane 6 to lane 5). Similarly,
SFPQ immunoprecipitated not only HEXIM1 and PSPC1 but also
DNAPKc and Ku70. Only the interactions with SFPQ and PSPC1
resisted RNase treatment (compare lane 8 to lane 7). Taken
together, our results suggest that the above-mentioned proteins
form a single RNP complex, independent of the 7SK snRNP/
P-TEFb complex and composed of three independently stable
modules, HEXIM1, DNA-PK, and paraspeckle components.
To confirm the existence of this new multimeric HEXIM1-con-
taining complex, we first performed glycerol gradient sedimenta-
tion of FLAG-eHEXIM1 immunoprecipitate. The presence of
eHEXIM1-interacting proteins in collected fractions was
analyzed by western blot (Figure 2A). As expected, eHEXIM1,
CyclinT1, CDK9, LARP7, and 7SK RNA co-migrated, with peak
intensity between fractions 13 and 17 (Figure 2A). However,
DNAPKc, Ku70, SFPQ, NONO, and PSPC1 co-sedimented
with HEXIM1, with peak intensity between fractions 9 and 13.
This suggests the presence of at least two distinct types of
 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

A B C
NE Flag-IP
Flag/HA-IP
eHEXIM1: - + + - + +
eHEXIM1 : - + RNase: - - + - - +

-DNAPKc Protein Unique % Protein eHEXIM1

Symbol Peptides coverage
200 - HEXIM1 167 55,99 CycT1
HEXIM2 22 44,41
-SFPQ, MATRIN3 CycT1 102 38,02 CDK9
116 -
CycT2 12 16,71
-CycT1, MEPCE,
96 - CDK9 53 36,29 Ku70
Ku80
LARP7 94 65
-LARP7, Ku70,
66 - PSPC1, RBM14 MEPCE 34 28,16 Ku80
DNAPKc 33 8,72
DNAPKc
55 - - eHEXIM1 KU70 8 10,25
KU80 9 7,06
-NONO SFPQ
SFPQ 17 19,66
NONO 17 38,43
NONO
PSPC1 8 18,36
37 - -CDK9, SRSF2
RBM14 5 8,97
PSPC1
31 - MATRIN3 12 16,77
RIF1

MW 1 2 1 2 3 4 5 6
(Kd)

Figure 1. Identification of eHEXIM1 Nuclear Partners

(A) FLAG-HA epitope-tagged HEXIM1 (eHEXIM1) was sequentially purified with anti-FLAG and anti-HA antibodies from nuclear extracts prepared from HeLa S3
mock cells (lane 1) or stably expressing eHEXIM1 (lane 2). Bound proteins were separated by SDS-PAGE and visualized by silver staining. Cellular nuclear
partners of eHEXIM1 were determined by tandem mass spectrometry. Major previously described HEXIM1 partners are indicated in black and identified partners
are indicated in red.
(B) Number of unique peptides and percentage of protein coverage of eHEXIM1 partners identified by tandem mass spectrometry (MS/MS).
(C) eHEXIM1 partners depend on RNA for their interaction. Anti-FLAG beads were used to immunoprecipitate eHEXIM1 and its interacting proteins from nuclear
extract. Lanes indicated in each panel show HeLa S3 control cells and cells overexpressing eHEXIM1, in the absence or presence of RNase, as indicated. The
various partners indicated were revealed by western blot using the specific antibodies shown.
See also Figure S1.

complexes containing HEXIM1. Next, we performed reciprocal
immunoprecipitation (reIP) experiments. NEs prepared from
HeLa S3 and HeLa S3eHEXIM1 were first depleted of CyclinT1
to remove P-TEFb complexes prior to eHEXIM1 IP. As shown
in Figure 2B, while CDK9 and HEXIM1 were recovered in
CyclinT1 IP, DNAPKc, Ku70, SFPQ, and PSPC1 were not
(compare lanes 4 and 5 to lane 3). Accordingly, analyses of the
flow-through extracts after CyclinT1 immunoprecipitation re-
vealed that depletion of CyclinT1 resulted in co-depletion of
CDK9, which reduced the levels of HEXIM1 but had no effect
on the levels of DNAPKc, Ku70, SFPQ, or PSPC1 (compare
lanes 7 and 8 to lanes 1 and 2; Figure S2A, compare lane 3 to
lane 2). Thus, CyclinT1 does not interact with DNAPKc, Ku70,
SFPQ, or PSPC1. These experiments reinforce the hypothesis
that HEXIM1 interaction with the identified partners is P-TEFb
independent.
CyclinT1-depleted extracts were subjected to FLAG-
eHEXIM1 immunoprecipitation, and bound material was eluted
using FLAG peptide. The eluate was subjected to immunopre-
cipitation a further time (reIP) using anti-HEXIM1-, anti-Ku70-,
anti-SFPQ-specific antibodies and IgG as a control. As ex-
pected, FLAG-eHEXIM1 immunoprecipitated Ku70, DNAPKc,
PSPC1, and SFPQ, but not CDK9 (Figure 2B, compare lane 12
to lane 11; Figure S2A, compare lane 5 to lane 6). The DNA-PK
component Ku70 immunoprecipitated HEXIM1, DNAPKc,
SFPQ, and PSPC1 (lane 15). Reciprocally, the paraspeckle com-
ponent SFPQ immunoprecipitated HEXIM1, Ku70, DNAPKc, and
PSPC1 (lane 16). Thus, these reIP experiments show that
HEXIM1, the DNA-PK complex, SFPQ, and PSPC1 associate
in a single complex that is independent of the previously charac-
terized HEXIM1/P-TEFb complex. We refer to this complex as
HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein
complex (HDP-RNP).
We next sought to determine the HEXIM1 interaction domains
involved in binding the DNA-PK and the paraspeckle subunits.
For this, we used well-characterized HEXIM1 deletion mutants
and an RNA-binding mutant (Michels et al., 2004) in coIP exper-
iments. Wild-type (WT) and HEXIM1 mutants were transfected
into HeLa cells and FLAG-immunoprecipitated from whole-cell
extracts (WCEs). The presence of P-TEFb and HDP-RNP sub-
units was assessed by western blotting (Figure S2B). As previ-
ously shown (Michels et al., 2004), the HEXIM1 (1180) fragment

Molecular Cell 67, 113, August 3, 2017 3

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

A 7SK-qRT-PCR
10% 40%
S3 eHEXIM1 (Fr) 1 3 5 7 9 11 13 15 17 19 21 23 100
nuclear extract eHEXIM1

CycT1 80

Percent max signal

Flag-IP CDK9
LARP7 60

DNAPKc
Glycerol gradient 40
sedimentation Ku70
SFPQ 20
NONO
7SK 0
Western PSPC1
blotting analysis qRT-PCR (Fr) 2 4 6 8 10 12 14 16 18 20 22 24

B
IP FT Input Re-IP

HEXIM1
Input IgG CycT1 IgG CycT1 Cyct1 depleted Flag-IP

SFPQ
Ku70
IgG
S3 eHEXIM1 eHEXIM1: - + + - + + - + - + - +
nuclear extract
CycT1 HEXIM1

Cyct1 IP
CDK9 DNAPKc

Unbound Ku70
HEXIM1
proteins
(FT)
DNAPKc SFPQ
Western
blotting Flag-IP Ku70 PSPC1
analysis (eHEXIM1)
SFPQ
-IgG

PSPC1 CDK9

Re-IP (HEXIM1, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Ku70,SFPQ)

7SKsnRNP HDP-RNP
MEPCE

SFPQ PSPC1
CycT1 HEXIM1 NONO
HEXIM1
CDK9
DNA-PK
7SK RNA
LARP7 ?

Figure 2. HEXIM1 Forms a 7SK RNP-Independent Ribonucleoprotein Complex Containing DNA-PK and Paraspeckle Subunits: The HEXIM1
DNA-PK Paraspeckle-RNP
(A) Left panel: experimental scheme. Middle panel: FLAG-purified eHEXIM1-associated complexes were separated by centrifugation through a 10%40%
glycerol gradient. Collected fractions were analyzed by western blot using the indicated antibodies. Right panel: RNA from even-numbered fractions was purified
and subjected to qRT-PCR analysis using 7SK-specific primers. The results are represented as a percentage relative to the maximal signal.
(B) Left panel: experimental scheme. Middle panel: nuclear extracts from HeLa S3 mock or eHEXIM-transfected cells were subjected to CyclinT1 immuno-
precipitation (CycT1 IP). Input as well as flow-through (FT) from CycT1 IP were probed with the indicated antibodies (lane 18). Right panel: FLAG-purified
eHEXIM1 from CycT1-depleted nuclear extract (lanes 11 and 12) was subjected to reciprocal immunoprecipitation (reIP) using HEXIM1-, Ku70-, or SFPQ-specific
antibodies or irrelevant IgG (lanes 1316). The presence of HDP-RNP complex subunits in FLAG-IP and reIPs was assessed by western blotting. Schematic
representation of the two HEXIM1 RNPs is shown at the bottom.
See also Figure S2.

4 Molecular Cell 67, 113, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020
did not interact with P-TEFb subunits, while the 181359 and
150359 fragments did (Figure S2B, compare lane 9 to lane 8
and lanes 10 and 11 to lane 8). HEXIM1 (181359) did not interact
with HDP-RNP subunits, while HEXIM1 (150359) retained the
interaction with the DNA-PK and lost the interaction with SFPQ
and PSPC1. These data therefore demonstrate that DNA-PK
binds to HEXIM1 amino acids (aa) 150181 and that paraspeckle
subunits bind to a region corresponding to aa 1150. As ex-
pected from the RNase treatment (in Figure 1C), the HEXIM1
ILAA RNA-binding mutant did not interact with P-TEFb nor with
the HDP-RNP subunits (compare lane 12 to lane 8). This exper-
iment also therefore shows that P-TEFb and HDP-RNP subunits
bind to distinct domains of HEXIM1 (the N and C termini, respec-
tively) and that HEXIM1 is the platform for the assembly of the
HDP-RNP (Figure S2C).
Taken together, these experiments show that HEXIM1 forms
two distinct RNP complexes, the 7SK snRNP/P-TEFb complex
and the 7SK RNA-independent HDP-RNP.
The Long Non-coding RNA NEAT1 Is Required for
HDP-RNP Assembly
To identify the RNA required for HDP-RNP assembly, we per-
formed IP/reIP experiments to purify total eHEXIM1-, inactive
P-TEFb-, and HDP-RNP-bound RNAs. ReIP using IgG was
used as a control (Figure 3A). RNA was isolated from the different
samples and subjected to RNA sequencing (RNA-seq). The
eHEXIM1 IP contained all the RNA species found in eHEXIM1/
CyclinT1 (inactive P-TEFb) and eHEXIM1/Ku70 (HDP-RNP) IPs
(Figure S3A). While the 7SK RNA was the major RNA found in
eHEXIM1/CyclinT1 IP, it was absent in eHEXIM1/Ku70 IP. In
contrast, the long non-coding RNA NEAT1, a structural compo-
nent of the paraspeckles (Clemson et al., 2009; Sasaki et al.,
2009), was highly enriched in eHEXIM1/Ku70 IP and absent in
eHEXIM1/CyclinT1 IP. Both 7SK and NEAT1 RNAs were highly
represented in eHEXIM1 IP and absent in the reIP using IgG.
To confirm these results, NEAT1 and 7SK RNA were quantified
by qRT-PCR in the reIPs (Figure 3B, right panel). 7SK RNA was
exclusively recovered in eHEXIM1/CyclinT1 reIP and NEAT1
RNA was recovered only in the eHEXIM1/Ku70 reIP. Both
RNAs were recovered in eHEXIM1 IP and absent in the IgG
control.
Next, we quantified NEAT1 in the fractions collected from the
glycerol gradient sedimentation of FLAG-purified eHEXIM1
shown in Figure 2A (Figure 3C). NEAT1 RNA was present in
the collected fractions with maximum intensity between frac-
tions 6 and 12, away from 7SK RNA. NEAT1 was present in
the fractions corresponding to HDP-RNP and absent in 7SK
snRNP/P-TEFb complex, suggesting that NEAT1 is the RNA
associated with HDP-RNP and required for its assembly. To
test this hypothesis, we analyzed the effect of 7SK and NEAT1
RNA knockdowns on the assembly of both inactive P-TEFb
and HDP-RNP. Knockdown efficiency of 7SK and NEAT1
RNAs was quantified by qRT-PCR (Figure S3B). While knock-
down of 7SK RNA resulted in loss of CyclinT1, it did not affect
the binding of HDP-RNP subunits to eHEXIM1 (Figure 3D,
compare lane 5 to lanes 4 and 2). Conversely, knockdown of
NEAT1 RNA resulted in loss of HDP-RNP subunits in eHEXIM1
IP with no effect on CyclinT1 binding (Figure 3D, compare
lane 6 to lanes 4 and 2). Interestingly, the HEXIM1 ILAA RNA-
binding mutant, which was impaired in P-TEFb 7SK RNA
binding and subunits of the HDP-RNP (Figure S2B, lane 12; Fig-
ure S3B, compare lane 7 to lane 9), failed to bind NEAT1 RNA
(Figure S3C, lower panel).
Taken together, these experiments show that NEAT1 is the
RNA associated with and required for the assembly of the
HDP-RNP and that HEXIM1 binds directly to NEAT1. Accord-
ingly, we found that, like SFPQ, both HEXIM1 and Ku70 partially
co-localized with the paraspeckle marker NEAT1 (Figure 3E;
Movies S1, S2, and S3).
The HDP-RNP Regulates the Innate Immune Response
DNA-PK acts as a DNA sensor and inducer of innate immune
activation through IRF3, TBK1, and the adaptor protein STING
(Ferguson et al., 2012). Herpes simplex virus infection may
lead to excessive formation of paraspeckles through enhanced
expression of NEAT1 (Imamura et al., 2014). We thus hypothe-
sized a potential role for the HDP-RNP in innate immune
responses. We analyzed the effect of knocking down the
HDP-RNP subunits on activation of type1 IFN by interferon stim-
ulatory DNA (ISD) and Poly(I:C) RNA. Knockdown efficiency of
the targeted subunits is shown in Figure S4. Both ISD and
Poly (I:C) RNA induced IFNa, IFNb, and the IFN response
gene MXA in control siRNA-transfected cells (siSCR) (Figure 4A).
Knockdown of the HDP-RNP subunits HEXIM1, Ku70, SFPQ,
and NEAT1 resulted in the loss of ISD-mediated IFNa, IFNb,
and MXA induction, but it had no effect on Poly(I:C)-induced re-
sponses. Moreover, knocking down the HDP-RNP subunits had
no effect on IFNa, IFNb, and MXA expression when cells were
treated with IFNa (Figure 4A). This suggests a role for HDP-
RNP in ISD-mediated signaling. In support of this hypothesis,
knockdown of HDP-RNP subunits impaired ISD-mediated
phosphorylation of IRF3 and its upstream regulator TBK1
without affecting their expression levels (Figure 4A, compare
lanes 6, 10, 18, and 22 to lanes 2 and 14). Knockdown of the
HDP-RNP subunits had no effect on phosphorylation of IRF3
and TBK1 induced by Poly(I:C) (compare lanes 7, 11, 19, and
23 to lanes 3 and 15). The observed effect of HDP-RNP on
ISD-mediated activation of IRF3 and TBK1 was not due to
down-modulation of the known DNA sensor cGAS or its adaptor
STING (Figure S4). Taken together, these experiments show
that the HDP-RNP acts as a positive regulator of DNA-mediated
IFN response gene induction upstream of IRF3 and TBK1
phosphorylation.
DNA-mediated activation of the cGAS sensor results in the
synthesis of a cGAMP from ATP and GTP (Li et al., 2013; Sun
et al., 2013). cGAMP binds to and activates the downstream
adaptor STING, initiating the activation pathway leading to the
production of type 1 IFN. To explore whether the HDP-RNP is
involved in the synthesis of cGAMP, we analyzed the effect of
HDP-RNP knockdown on cGAMP-mediated activation of IFN.
As shown in Figure 4B, knockdown of HDP-RNP subunits
impaired both ISD- and cGAMP-mediated induction of both
IFNb mRNA expression and IRF3 phosphorylation. Our data
thus demonstrate that HDP-RNP regulates the DNA-mediated
innate immune response upstream of IRF3 and TBK1 phosphor-
ylation and downstream of cGAMP synthesis.
Molecular Cell 67, 113, August 3, 2017 5
 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

S3 eHEXIM1 B
A nuclear extract
CycT1 IP

100
Unbound proteins
(FT)

% of RNA relative to eHEXIM1

80

Flag-IP Flag-IP
60

bound RNA
Elution Elution

40
eHEXIM1 eHEXIM1 pTEFb
associated partners independent partners
20

CycT1 Re-IP IgG Re-IP Ku70 Re-IP

0

IgG

IgG
HEXIM1

Ku70

HEXIM1

Ku70
CycT1

CycT1
RNA purification and sequencing

7SK NEAT 1

eHEXIM1 eHEXIM1-pTEFb IgG eHEXIM1-DNAPK

associated RNA associated RNA associated RNA associated RNA

C D Flag-IP

eHEXIM1 : - + + + + +
7SK NEAT1
RNAse : - - + - - -
100
siSCR : - - - + - -
si7SK : - - - - + - HDP RNP
80
Percent max signal

siNEAT1 : - - - - - +

60 eHEXIM1 SFPQ PSPC1

CycT1 HEXIM1 NONO
40
DNAPKc
DNAPK
20 Ku70
NEAT1 RNA
SFPQ
0
(Fr) 2 4 6 8 10 12 14 16 18 20 22 24 1 2 3 4 5 6

(legend on next page)

6 Molecular Cell 67, 113, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

A 80 MXA Figure 4. The HDP-RNP Is Involved in the

75 cGAS-STING-IRF3-Dependent Innate Im-
70 mune Response to ISD
65
(A) Upper panel: HeLa cells were transfected with
60
55 siRNA targeting HEXIM1, Ku70, NEAT1, SFPQ, or a
50 non-targeting siRNA (SCR). Transfected cells were
Fold induction

45 mock-treated (NS) or treated for 6 hr with 10 mg/mL

40
ISD or 1 mg/mL Poly(I:C) or 100 IU/mL IFNa. The
35
30 levels of induction of IFNa, IFNb, and MXA mRNAs
25 were measured by qRT-PCR. The graph shows
20 mean SD (n = 3) of the fold induction relative to the
15
control condition (siRNA SCR NS). Lower panel:
10
5 whole-cell extracts were analyzed by western blot-
0 ting using the indicated antibodies.
NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN (B) Upper panel: experiment performed as in (A)
siSCR siHEXIM1 siKu70 siNEAT1 siSFPQ except that siRNA-transfected cells were mock-
treated (NS) or treated for 6 hr with 10 mg/mL ISD or
siSCR siHEXIM1 siNEAT1 siSCR siKu70 siSFPQ 20 -30 cGAMP. Induction of IFNb was measured by
NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN NS ISD I:C IFN qRT-PCR. The graph shows mean SD (n = 2) of the
pIRF3 pIRF3
fold induction relative to the control condition
IRF3 IRF3 (siSCR NS). Lower panel: whole-cell extracts from
the experiment performed in the upper panel were
pTBK1 pTBK1
analyzed by western blotting using the indicated
TBK1 TBK1 antibodies.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 See also Figure S4.

B
35

30
and IRF3. Thus, the presence of cGAS siSCR siHEXIM1 siNEAT1
25
and IRF3 in eHEXIM1 IPs used in Figure 1
Fold induction

NS ISD 2-3 NS ISD 2-3 NS ISD 2-3

20 was analyzed. Both cGAS and IRF3 were
pIRF3

15 present in eHEXIM1 IP, and these interac-

IRF3
10
tions were lost upon RNase treatment (Fig-
1 2 3 4 5 6 7 8 9
ure S5A). Interestingly, IP of endogenous
5 siSCR siKu70 siSFPQ
HEXIM1 and Ku70 also recovered cGAS
NS ISD 2-3 NS ISD 2-3 NS ISD 2-3
0 in an RNA-dependent manner (Figure S5B).
ISD

ISD

NS
ISD

ISD

NS
ISD
NS

NS

NS
2'-3' cGAMP

2'-3' cGAMP

2'-3' cGAMP

2'-3' cGAMP

2'-3' cGAMP

pIRF3
To gain insight into the regulation of the
IRF3 HDP-RNP complex, we asked whether
11 12 13 14 15 16 17 18 19 ISD may remodel the composition of the
siSCR siHEXIM1 siKu70 siSFPQ siNEAT1 HDP-RNP and its interaction with the
signaling pathway leading to IFN induction.
For this purpose, we performed a sequen-
Interferon Stimulatory DNA Regulates the Assembly and tial immunoprecipitation (IP/reIP) experiment, as described in
Composition of the HDP-RNP Figure 2B, using WCEs prepared from HeLa and HeLa eHEXIM1
Our biochemical and functional data define the HDP-RNP com- that were mock or ISD treated. As expected, immunoprobing of
plex as a key regulator of ISD-mediated innate immune activa- WCEs showed that ISD treatment had no effect on the expres-
tion. First, we questioned the possible interaction between the sion levels of cGAS, its partner PQBP1, IRF3, or the RNA sensor
HDP-RNP and mediators of innate immune activation cGAS RIG-I. As expected, ISD enhanced the expression of STING and

Figure 3. NEAT1 Is an Integral Component of the HDP-RNP Complex

(A) Left panel: experimental scheme used to purify the RNAs associated with the HDP-RNP complex. eHEXIM1 (blue), eHEXIM1-P-TEFb (red), and eHEXIM1-
Ku70 (green) -associated RNAs were purified using IP and reIP as depicted. Irrelevant IgG antibody was used as control (black). Purified RNAs were sequenced.
(B) NEAT1 and 7SK RNA were quantified in the different IPs by qRT-PCR using specific primers. The graph shows, for each condition, the percentage of NEAT1 or
7SK RNAs relative to the RNAs bound to eHEXIM1 (blue).
(C) RNAs from even-numbered fractions of the glycerol gradient shown in Figure 2A were subjected to qRT-PCR analysis using 7SK- and NEAT1-specific primers.
(D) HeLa S3 overexpressing eHEXIM1 were transfected with siRNA targeting 7SK or NEAT1 RNAs (lanes 46); a non-targeting siRNA (siSCR, lane3) was used as a
control. Whole-cell extracts were then prepared, treated with RNase or not (lanes 13), and subjected to FLAG IP. FLAG-IPs were analyzed by western blotting
using the indicated antibodies. A schematic representation of the HDP-RNP complex containing the lncRNA NEAT1 is shown on the right.
(E) The localization of NEAT1 RNA and HDP subunits was assessed by immuno-fluorescence in situ hybridization (FISH) in HeLa cells. NEAT1 foci (red, column 3)
was detected by RNA-FISH using specific probes, and Ku70 (upper panel), HEXIM1 (middle panel), and SFPQ (lower panel) were detected by immunostaining
using specific antibodies (green, column 2). Nuclei were stained with DAPI (blue, column 1). Merged images are shown in column 4.
See also Figure S3 and Movies S1, S2, and S3.

Molecular Cell 67, 113, August 3, 2017 7

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

A induced the phosphorylation of IRF3 (Figure 5A, lanes 13).

Input Flag-IP Re-IP
While the levels of HDP-RNP subunits were not affected by

HEXIM1
ISD, an activation of DNAPKc was observed, as witnessed by

SFPQ
Ku70
IgG
enhanced phosphorylation at serine 2,056 (Figure 5A, lanes
eHEXIM1: - + + - + + + + + + + + + 13). When eHEXIM1 and its associated partners were purified
ISD: - - + - - + - - + - + - + from mock- or ISD-treated WCEs, we found a constitutive bind-
HEXIM1 ing of Ku70 and DNAPKc, but a loss of the paraspeckle proteins
Ku70 SFPQ and PSPC1, upon ISD treatment (Figure 5A, compare lane
6 to lane 5). cGAS and its partners PQBP1, STING, and IRF3
DNAPKc
associated with eHEXIM1 upon ISD treatment, which also
pDNAPKc-Ser2056
caused STING to be recruited to the HDP-RNP (Figure 5A,
SFPQ compare lane 6 to lane 5). ISD treatment also induced the phos-
phorylation of DNAPKc and of IRF3 associated with HEXIM1. Of
PSPC1
note, eHEXIM1 did not interact with the RNA sensor RIG-I. Alto-
STING
gether, these experiments not only revealed an interaction
cGAS among HEXIM1, cGAS, PQBP1, STING, and IRF3 but also
PQBP1 showed that HEXIM1s interactions with its partners are regu-
lated upon innate immune activation by ISD.
RIG-I
To assess whether the remodeling of HEXIM1s interactions
pIRF3 by ISD occurred within the HDP-RNP, we performed reIP on
IRF3 the eHEXIM1-bound proteins using HEXIM1-, KU70-, or SFPQ-
Actin specific antibodies (Figure 5A, lanes 713). Consecutive immu-
1 2 3 4 5 6 7 8 9 10 11 12 13 noprecipitation with HEXIM1 and Ku70 retained HEXIM1,
Ku70, DNAPKc, cGAS, PQBP1, and IRF3, whereas SFPQ and
PSPC1 were lost upon ISD treatment. We also observed an
B
enhanced recruitment of STING and phosphorylation of associ-
IP cGAS
IgG

Input ated DNAPKc and of IRF3 active forms (compare lane 9 to lane 8
Si SCR : + + - - + + + - - and lane 11 to lane 10). SFPQ reIP revealed the presence of the
Si HEXIM1 : - - + + - - - + + HDP-RNP subunits cGAS, PQBP1, STING, and IRF3 in mock
ISD : - + - + - - + - +
that was not treated with ISD (lane 12), but not upon treatment
cGAS
with ISD (compare lane 13 to lane 12). Taken together, these ex-
HEXIM1 periments show that the mediators of innate immune activation,
DNAPKc cGAS, PQBP1, STING, and IRF3, interact with the HDP-RNP.
Triggering the innate immune response by ISD results in the re-
pDNAPKc-Ser2056
modeling of the HDP-RNP complex and activation of both
Ku70 DNAPKc and IRF3. To further characterize the interaction be-
SFPQ tween the HDP-RNP and the mediators of the innate immune
PSPC1 response, cGAS was immunoprecipitated from WCEs prepared
from control or HEXIM1 knockdown cells that were mock or ISD
PQBP1
treated. The presence of HDP-RNP subunits and PQBP1,
STING STING, and IRF3 was analyzed by western blotting (Figure 5B).
pIRF3
Endogenous cGAS immunoprecipitated subunits of the HDP-
RNP complex PQBP1, STING, and IRF3 (compare lane 6 to
IRF3
lane 5). ISD treatment resulted in the loss of SFPQ, PSPC1 bind-
Actin
ing, and enhanced recruitment of STING without affecting the
1 2 3 4 5 6 7 8 9 binding of the other subunits (compare lane 7 to lane 6). ISD
treatment induced the activating phosphorylation of DNAPKc
and of IRF3 bound to cGAS. Interestingly, knockdown of
Figure 5. Regulation of the HDP-RNP Complex in Response to DNA
Stimulation
HEXIM1 stopped the HDP-RNP subunits binding to cGAS
(A) Western blots of whole-cell extracts prepared from HeLa eHEXIM1-over- (compare lane 8 to lane 6). Although, HEXIM1 knockdown did
expressing cells that were mock-treated or treated for 6 hr with interferon not affect cGAS interaction with PQBP1, STING, and IRF3, it
stimulatory DNA (10 mg/mL) (lanes 13). These extracts were subjected to abrogated the phosphorylation of cGAS-bound IRF3 induced
FLAG IP (lanes 46). The bulk of the FLAG-immunoprecipitated complexes by ISD (compare lane 9 to lane 7), suggesting that the HDP-RNP
(which are resolved in lanes 5 and 6) were subjected to reciprocal IPs (reIPs)
using HEXIM1-, Ku70-, and SFPQ-specific antibodies or irrelevant IgG
(lanes 713). The presence of HDP-RNP complex subunits in FLAG-IP and and subjected to endogenous cGas IP using a specific antibody. The presence
reIPs was assessed by western blot (WB) using the indicated antibodies. of HDP-RNP complex subunits in the whole-cell extracts and IPs was
(B) Whole-cell extracts prepared from HeLa cells transfected with siRNA tar- assessed by WB using the indicated antibodies.
geting HEXIM1 that were mock-treated or treated for 6 hr with ISD (10 mg/mL) See also Figure S5.

8 Molecular Cell 67, 113, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

A B 9

Fold induction after KSHV Infection

7

STING HEXIM1 6
Actin Actin
5

100 4
% NEAT1-RNA/ SCR
80
3
Ku70 60
2
Actin 40
1
20

0 0
siSCR siSTING siHEXIM1 siKu70 siNEAT1

C Input IP HEXIM1 IP MYC

IgG

IgG

Myc-ORF52 : - - + + + - - + + + + +
ISD : - + - + - - + - + - - +
HEXIM1

Myc-ORF52

cGas

PQBP1

CycT1

IRF3

pIRF3
1 2 3 4 5 6 7 8 9 10 11 12

Figure 6. The HDP-RNP Is Required for the KSHV-Mediated Innate Immune Response
(A) HUVECs were transfected with siRNA targeting STING (siSTING), HEXIM1 (siHEXIM1), Ku70 (siKu70), or a control non-specific siRNA (siSCR) for 48 hr or with
NEAT1 (siNEAT1) for 8 hr. Specific inhibitions were assessed by western blot for siSTING, siHEXIM1, and siKu70 and by qRT-PCR for siNEAT.
(B) Cells were then infected with KSHV (40 genome copies per cell). At 16 hr post-infection, cells were harvested, RNAs were purified, and IFNb expression was
quantified by qRT-PCR using specific primers and normalized to actin expression. Results are expressed as fold increased expression compared to non-infected
cells. Results are presented as means of a triplicate representative of at least two independent experiments.
(C) Whole-cell extracts prepared from HeLa transfected with Myc-ORF52 that were mock-treated or treated for 6 hr with ISD (10 mg/mL) were subjected to IP of
endogenous HEXIM1 (lanes 59) or MYC IP using specific antibodies. Input and bound complexes were analyzed by WB using the indicated antibodies.
See also Figure S6.

is required for IRF3 phosphorylation. Interestingly, knockdown of
Ku70 did not affect the binding of the HDP-RNP to cGAS,
PQBP1, or IRF3 (Figure S5C, compare lane 6 to lane 8). ISD treat-
ment of KU70 knockdown cells resulted in SFPQ and PSPC1
release. ISD treatment did not affect the interaction among
HEXIM1, DNAPKc and cGAS, PQBP1, and IRF3 nor the recruit-
ment of STING (compare lane 6 to lane 7). However, phosphor-
ylation of cGAS-bound DNAPKc and IRF3 was abrogated
(compare lane 8 to lane 9). These results highlight the role of
HEXIM1 as a platform for the assembly of the HDP-RNP with
the innate immune activating pathway and that Ku70 is required
for the ISD-induced activation of DNAPKc and IRF3 within the
HDP-RNP.
The HDP-RNP Complex Is Required for cGAS-
Dependent Kaposis Sarcoma-Associated Herpesvirus-
Mediated Activation of the Innate Immune Response
We next asked whether the HDP-RNP is involved in the cGAS-
mediated innate immune response against DNA viruses. To this
aim, we used Kaposis sarcoma-associated herpesvirus (KSHV)
infection of human umbilical vein endothelial cells (HUVECs),
which activates the cGAS-STING pathway (Ma et al., 2015). First,
using specific siRNA against HEXIM1, NEAT1, Ku70, and SFPQ,
we confirmed the requirement for the HDP-RNP for ISD-medi-
ated activation of IFNb in HUVECs (Figure S6). Next, we assessed
the ability of KSHV to induce innate immune response in
HUVECs. As shown in Figure 6, knockdown of HEXIM1, Ku70,

Molecular Cell 67, 113, August 3, 2017 9

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Basal State Activated State

dsDNA STING
STING
HDP-RNP STING STING HDP-RNP
STING STING

SFPQ PSPC1
cGAS HEXIM1 NONO DNA SENSING cGAS HEXIM1
P
DNA-PK P DNA-PK
STING
STING IRF3
SFPQ PSPC1
IRF3 STING
NONO
STING
STING NEAT1 RNA STING
STING
STING

P
IRF3 P
IRF3

TYPE-1 IFN GENES ACTIVATION

Figure 7. Proposed Model for the HDP-RNP Complex Interaction with the cGAS-STING-IRF3 Innate Immune Pathway and Its Regulation
upon DNA Stimulation

NEAT1, and STING results in inhibition of the KSHV-mediated
response, as analyzed by measuring IFNb mRNA. To circumvent
cGAS and STING activation, KSHV encodes vIRF1, which targets
STING and prevents its phosphorylation, and LANA and ORF52,
which target cGAS and inhibit its activation (Wu et al., 2015;
Zhang et al., 2016). Therefore, we explored the effect of ORF52
expression on the interaction between the HDP-RNP and
cGAS. Expression of ORF52 did not affect the expression levels
of HEXIM1, cGAS, PQBP1, and IRF3 (Figure 6C, lanes 14). As
previously described, expression of ORF52 prevented the
phosphorylation of IRF3 induced by ISD (compare lane 4 to
lane 2). Interestingly, expression of ORF52 resulted in the loss
of binding of cGAS and PQBP1 to HEXIM1, with no effect on
CyclinT1 binding (compare lanes 8 and 9 to lanes 6 and 7).
ORF52 IP recovered cGAS and PQBP1, but not HEXIM1
(compare lane 10 to lane 12). Overall, our data show that targeting
HEXIM1-cGAS interaction is a viral strategy to avoid the initiation
of innate immune responses.
DISCUSSION
In this study, we identified a HEXIM1-containing ribonuclear pro-
tein complex composed also of DNA-PK, paraspeckle subunits,
and the long non-coding RNA (lncRNA) NEAT1, which we termed
the HDP-RNP. This complex is independent of P-TEFb and the
7SK RNA. The P-TEFb constituent CycT1 does not interact
with any of the HDP-RNP subunits, and knockdown of 7SK
does not affect HDP-RNP assembly. Our biochemical data
show that HEXIM1 interacts with NEAT1 to create the necessary
platform for the assembly of the HDP-RNP. Knockdown of
HEXIM1 (data not shown) or NEAT1 results in loss of the interac-
tion between the DNA-PK and the paraspeckle components of
the HDP-RNP, which bind to distinct regions of HEXIM1. The
RNA-binding-defective mutant of HEXIM1 (ILAA) fails to bind
NEAT1 and to assemble the HDP-RNP.
Our functional data established HDP-RNP as a key regulator of
ISD- and KSHV-mediated activation of the cGAS-STING-IRF3
pathway. We found that endogenous cGAS, PQBP1, and IRF3
interact with the HDP-RNP. Upon stimulation with ISD, the
HDP-RNP is remodeled in its composition, with recruitment of
STING, release of SFPQ and PSPC1, and phosphorylation of
DNAPKc and IRF3 (Figure 7C). This is in agreement with previous
findings showing that DNA-PK is required for the IRF3-depen-
dent innate immune response against Vaccinia virus and that
DNA-PK subunits interact with IRF3 and STING (Ferguson
et al., 2012). Furthermore, we observed that Ku70 is required
for ISD-induced DNAPKc activation and IRF3 phosphorylation.
These observations open important questions about the role of
DNAPKc kinase activity in the remodeling of the HDP-RNP
and/or in the activation of cGAS, STING, TBK1, or IRF3. We
found that the HDP-RNP is involved downstream of DNA recog-
nition and upstream of IRF3 and TBK1 activation. However, we
cannot exclude the possibility that the HDP-RNP might also
regulate cGAS enzymatic activity. Further studies are necessary
to determine the precise function of each subunit of the HDP-
RNP in regulating the activation of the cGAS-STING-IRF3
pathway.
Sensing of nucleic acids has been mainly studied and reported
in the cytoplasm and in the endosomes of the cell (Barbalat et al.,
2011; Paludan and Bowie, 2013). However, recent evidence

10 Molecular Cell 67, 113, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020
suggests the existence of nuclear sensors involved in DNA
recognition (Diner et al., 2015). Nuclear DNA sensing might
represent an advantage for the cell at two levels. First, most
DNA viruses require nuclear entry for their replication, and their
genomes are highly protected during trafficking through the
cytoplasm by their capsid. For instance, the DNA sensor IFI16
senses herpes viral DNA in the nucleus of human primary fibro-
blasts, and it initiates STING and IRF3-dependent innate
immune signaling (Kerur et al., 2011; Orzalli et al., 2013). Interest-
ingly, cGAS localizes to the nucleus and may cooperate with
IFI16 (Orzalli et al., 2015). Our finding that the HDP-RNP, which
is also found in the nucleus, is required for KSHV-mediated acti-
vation of innate immune responses reinforces the concept of nu-
clear sensing. Second, replication stress and DNA damage
result in the accumulation of unresolved DNA that is recognized
by innate immune sensors, and they result in the production of
inflammatory cytokines that promote growth arrest or apoptosis.
In this regard, the presence of a DNA-sensing unit in the
nucleus could rapidly initiate detection of genome instability
and activate an inflammatory pathway that may favor cell sur-
vival and tissue reparation (Bregnard et al., 2016; Dufour et al.,
2003; Rathbun et al., 1997). Accordingly, DNA damage induction
in MCF7 cells results in activation of STING and its co-localiza-
tion with phosphorylated IRF3 and gH2AX in nuclear foci (Gaston
et al., 2016). Inactivation of STING decreased DNA damage-
induced inflammation, promoted cell death, and delayed colony
formation (Gaston et al., 2016). Interestingly, some subunits
of the HDP-RNP have been directly implicated in the DNA
damage repair (DDR), particularly in double-strand break (DSB)
repair (Adriaens et al., 2016; Salton et al., 2010; Udayakumar
and Dynan, 2015). NEAT1 and/or paraspeckles are required to
preserve genome integrity and to resolve DSBs (Adriaens
et al., 2016). It is therefore likely that the HDP-RNP constitutes
a fundamental platform at the crossroad of diverse danger
signals in the cell. Future investigations will help to determine
the role of the HDP-RNP in the innate response to other DNA vi-
ruses and in the interplay between DDR and the inflammatory
response.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell lines
d METHOD DETAILS
B Plasmids
B Virus production and cell line transduction
B Purification of HEXIM1-associated complexes
B Glycerol Gradient Sedimentation Analysis and Recip-
rocal Immunoprecipitations
B Reagents, transfection and siRNA
B RNaseq
B Immuno-FISH
B Whole cell extract preparation and IPs
B RNA extraction and RT-qPCR
B KSHV viral production
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, two tables, and three movies
and can be found with this article online at http://dx.doi.org/10.1016/j.
molcel.2017.06.020.
AUTHOR CONTRIBUTIONS
M.B. conceived the study. M.B., M.M., and A.C. designed experiments,
interpreted data, and wrote the manuscript. M.M., A.C., and Y.B. performed
experiments. R.R. performed the RNA-seq bioinformatics analyses. D.S. and
E.D. did the RNA-seq. J.C. performed the analyses of immunofluorescent
imaging. S.A. and O.S. performed experiments. All the authors discussed
the data.
ACKNOWLEDGMENTS
We thank members of the Molecular Virology lab for critical reading of the
manuscript. We thank Oliver Bensaude for the kind gift of pAdRSV-FLAG con-
taining the HEXIM1 constructs and Thomas Schulz for the pCDNA4-MYC
containing KSHV ORF52. We are grateful to Montpellier RIO Imaging for
help with the microscopy analyses. RNA-seq experiments were performed
using MGX-Montpellier facilities. This work was supported by grants from
the ANRS, European FP7 HIT HIDDEN HIV contract 305762 and
MSDavenir. M.M. was supported by European FP7 HIT HIDDEN HIV con-
tract 305762 and by foundation Bettencourt Schueller and A.C. was sup-
ported by SIDACTION.
Received: January 18, 2017
Revised: May 15, 2017
Accepted: June 16, 2017
Published: July 13, 2017
REFERENCES
Ablasser, A., Schmid-Burgk, J.L., Hemmerling, I., Horvath, G.L., Schmidt, T.,
Latz, E., and Hornung, V. (2013). Cell intrinsic immunity spreads to bystander
cells via the intercellular transfer of cGAMP. Nature 503, 530534.
Adriaens, C., Standaert, L., Barra, J., Latil, M., Verfaillie, A., Kalev, P., Boeckx,
B., Wijnhoven, P.W.G., Radaelli, E., Vermi, W., et al. (2016). p53 induces forma-
tion of NEAT1 lncRNA-containing paraspeckles that modulate replication
stress response and chemosensitivity. Nat. Med. 22, 861868.
Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeqa Python framework to
work with high-throughput sequencing data. Bioinformatics 31, 166169.
Barbalat, R., Ewald, S.E., Mouchess, M.L., and Barton, G.M. (2011). Nucleic
acid recognition by the innate immune system. Annu. Rev. Immunol. 29,
185214.
Bregnard, C., Guerra, J., Dejardin, S., Passalacqua, F., Benkirane, M., and
Laguette, N. (2016). Upregulated LINE-1 activity in the Fanconi anemia cancer
susceptibility syndrome leads to spontaneous pro-inflammatory cytokine pro-
duction. EBioMedicine 8, 184194.
Burdette, D.L., and Vance, R.E. (2013). STING and the innate immune
response to nucleic acids in the cytosol. Nat. Immunol. 14, 1926.
Burdette, D.L., Monroe, K.M., Sotelo-Troha, K., Iwig, J.S., Eckert, B., Hyodo,
M., Hayakawa, Y., and Vance, R.E. (2011). STING is a direct innate immune
sensor of cyclic di-GMP. Nature 478, 515518.
Clemson, C.M., Hutchinson, J.N., Sara, S.A., Ensminger, A.W., Fox, A.H.,
Chess, A., and Lawrence, J.B. (2009). An architectural role for a nuclear non-
coding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol.
Cell 33, 717726.
Molecular Cell 67, 113, August 3, 2017 11
 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020
Dai, P., Wang, W., Cao, H., Avogadri, F., Dai, L., Drexler, I., Joyce, J.A., Li,
X.-D., Chen, Z., Merghoub, T., et al. (2014). Modified vaccinia virus Ankara
triggers type I IFN production in murine conventional dendritic cells via
a cGAS/STING-mediated cytosolic DNA-sensing pathway. PLoS Pathog. 10,
e1003989.
Davis, A.J., Chen, B.P., and Chen, D.J. (2014). DNA-PK: a dynamic enzyme in a
versatile DSB repair pathway. DNA Repair (Amst.) 17, 2129.
Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. (1983). Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated mammalian
nuclei. Nucleic Acids Res. 11, 14751489.
Diner, B.A., Lum, K.K., and Cristea, I.M. (2015). The emerging role of nuclear
viral DNA sensors. J. Biol. Chem. 290, 2641226421.
Diribarne, G., and Bensaude, O. (2009). 7SK RNA, a non-coding RNA regu-
lating P-TEFb, a general transcription factor. RNA Biol. 6, 122128.
Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut,
P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq
aligner. Bioinformatics 29, 1521.
Dufour, C., Corcione, A., Svahn, J., Haupt, R., Poggi, V., Bekassy, A.N.,
Scime, R., Pistorio, A., and Pistoia, V. (2003). TNF-a and IFN-g are overex-
pressed in the bone marrow of Fanconi anemia patients and TNF-a sup-
presses erythropoiesis in vitro. Blood 102, 20532059.
Ferguson, B.J., Mansur, D.S., Peters, N.E., Ren, H., and Smith, G.L. (2012).
DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. eLife 1,
e00047.
Fox, A.H., and Lamond, A.I. (2010). Paraspeckles. Cold Spring Harb. Perspect.
Biol. 2, a000687.
Gao, D., Wu, J., Wu, Y.-T., Du, F., Aroh, C., Yan, N., Sun, L., and Chen, Z.J.
(2013). Cyclic GMP-AMP synthase is an innate immune sensor of HIV and
other retroviruses. Science 341, 903906.
Gaston, J., Cheradame, L., Yvonnet, V., Deas, O., Poupon, M.-F., Judde,
J.-G., Cairo, S., and Goffin, V. (2016). Intracellular STING inactivation sen-
sitizes breast cancer cells to genotoxic agents. Oncotarget 7, 77205
77224.
Gentili, M., Kowal, J., Tkach, M., Satoh, T., Lahaye, X., Conrad, C., Boyron, M.,
Lombard, B., Durand, S., Kroemer, G., et al. (2015). Transmission of innate im-
mune signaling by packaging of cGAMP in viral particles. Science 349,
12321236.
Hernandez, C., Huebener, P., and Schwabe, R.F. (2016). Damage-associ-
ated molecular patterns in cancer: a double-edged sword. Oncogene 35,
59315941.
Imamura, K., Imamachi, N., Akizuki, G., Kumakura, M., Kawaguchi, A., Nagata,
K., Kato, A., Kawaguchi, Y., Sato, H., Yoneda, M., et al. (2014). Long noncod-
ing RNA NEAT1-dependent SFPQ relocation from promoter region to
paraspeckle mediates IL8 expression upon immune stimuli. Mol. Cell 53,
393406.
Ishikawa, H., and Barber, G.N. (2008). STING is an endoplasmic reticu-
lum adaptor that facilitates innate immune signalling. Nature 455,
674678.
Kerur, N., Veettil, M.V., Sharma-Walia, N., Bottero, V., Sadagopan, S., Otageri,
P., and Chandran, B. (2011). IFI16 acts as a nuclear pathogen sensor to induce
the inflammasome in response to Kaposi Sarcoma-associated herpesvirus
infection. Cell Host Microbe 9, 363375.
Lam, E., Stein, S., and Falck-Pedersen, E. (2014). Adenovirus detection by the
cGAS/STING/TBK1 DNA sensing cascade. J. Virol. 88, 974981.
Lew, Q.J., Chia, Y.L., Chu, K.L., Lam, Y.T., Gurumurthy, M., Xu, S., Lam, K.P.,
Cheong, N., and Chao, S.H. (2012). Identification of HEXIM1 as a positive regu-
lator of p53. J. Biol. Chem. 287, 3644336454.
Li, Q., Cooper, J.J., Altwerger, G.H., Feldkamp, M.D., Shea, M.A., and Price,
D.H. (2007). HEXIM1 is a promiscuous double-stranded RNA-binding protein
and interacts with RNAs in addition to 7SK in cultured cells. Nucleic Acids
Res. 35, 25032512.
12 Molecular Cell 67, 113, August 3, 2017
Li, X.-D., Wu, J., Gao, D., Wang, H., Sun, L., and Chen, Z.J. (2013). Pivotal roles
of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects.
Science 341, 13901394.
Ma, Z., and Damania, B. (2016). The cGAS-STING Defense Pathway and Its
Counteraction by Viruses. Cell Host Microbe 19, 150158.
Ma, Z., Jacobs, S.R., West, J.A., Stopford, C., Zhang, Z., Davis, Z., Barber,
G.N., Glaunsinger, B.A., Dittmer, D.P., and Damania, B. (2015). Modulation
of the cGAS-STING DNA sensing pathway by gammaherpesviruses. Proc.
Natl. Acad. Sci. USA 112, E4306E4315.
Michels, A.A., Nguyen, V.T., Fraldi, A., Labas, V., Edwards, M., Bonnet, F.,
Lania, L., and Bensaude, O. (2003). MAQ1 and 7SK RNA interact with
CDK9/cyclin T complexes in a transcription-dependent manner. Mol. Cell.
Biol. 23, 48594869.
Michels, A.A., Fraldi, A., Li, Q., Adamson, T.E., Bonnet, F., Nguyen, V.T.,
Sedore, S.C., Price, J.P., Price, D.H., Lania, L., and Bensaude, O. (2004).
Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/
cyclin T) inhibitor. EMBO J. 23, 26082619.
Nakagawa, S., and Hirose, T. (2012). Paraspeckle nuclear bodiesuseful use-
lessness? Cell. Mol. Life Sci. 69, 30273036.
Nakatani, Y., and Ogryzko, V. (2003). Immunoaffinity purification of mamma-
lian protein complexes. Methods Enzymol. 370, 430444.
Orzalli, M.H., Conwell, S.E., Berrios, C., DeCaprio, J.A., and Knipe, D.M.
(2013). Nuclear interferon-inducible protein 16 promotes silencing of herpesvi-
ral and transfected DNA. Proc. Natl. Acad. Sci. USA 110, E4492E4501.
Orzalli, M.H., Broekema, N.M., Diner, B.A., Hancks, D.C., Elde, N.C., Cristea,
I.M., and Knipe, D.M. (2015). cGAS-mediated stabilization of IFI16 promotes
innate signaling during herpes simplex virus infection. Proc. Natl. Acad. Sci.
USA 112, E1773E1781.
Paludan, S.R. (2015). Activation and regulation of DNA-driven immune re-
sponses. Microbiol. Mol. Biol. Rev. 79, 225241.
Paludan, S.R., and Bowie, A.G. (2013). Immune sensing of DNA. Immunity 38,
870880.
Qian, C., Liu, J., and Cao, X. (2014). Innate signaling in the inflammatory im-
mune disorders. Cytokine Growth Factor Rev. 25, 731738.
Rathbun, R.K., Faulkner, G.R., Ostroski, M.H., Christianson, T.A., Hughes, G.,
Jones, G., Cahn, R., Maziarz, R., Royle, G., Keeble, W., et al. (1997).
Inactivation of the Fanconi anemia group C gene augments interferon-
gamma-induced apoptotic responses in hematopoietic cells. Blood 90,
974985.
Salton, M., Lerenthal, Y., Wang, S.-Y., Chen, D.J., and Shiloh, Y. (2010).
Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response.
Cell Cycle 9, 15681576.
Sasaki, Y.T.F., Ideue, T., Sano, M., Mituyama, T., and Hirose, T. (2009).
MENepsilon/beta noncoding RNAs are essential for structural integrity of nu-
clear paraspeckles. Proc. Natl. Acad. Sci. USA 106, 25252530.
Shimizu, N., Ouchida, R., Yoshikawa, N., Hisada, T., Watanabe, H., Okamoto,
K., Kusuhara, M., Handa, H., Morimoto, C., and Tanaka, H. (2005). HEXIM1
forms a transcriptionally abortive complex with glucocorticoid receptor
without involving 7SK RNA and positive transcription elongation factor b.
Proc. Natl. Acad. Sci. USA 102, 85558560.
Stetson, D.B., and Medzhitov, R. (2006). Type I interferons in host defense.
Immunity 25, 373381.
Sun, L., Wu, J., Du, F., Chen, X., and Chen, Z.J. (2013). Cyclic GMP-AMP syn-
thase is a cytosolic DNA sensor that activates the type I interferon pathway.
Science 339, 786791.
Tanaka, Y., and Chen, Z.J. (2012). STING specifies IRF3 phosphorylation by
TBK1 in the cytosolic DNA signaling pathway. Sci. Signal. 5, ra20.
Udayakumar, D., and Dynan, W.S. (2015). Characterization of DNA binding
and pairing activities associated with the native SFPQ$NONO DNA repair pro-
tein complex. Biochem. Biophys. Res. Commun. 463, 473478.
 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Wu, J.J., Li, W., Shao, Y., Avey, D., Fu, B., Gillen, J., Hand, T., Ma, S., Liu, X.,
Miley, W., et al. (2015). Inhibition of cGAS DNA Sensing by a Herpesvirus Virion
Protein. Cell Host Microbe 18, 333344.
Yik, J.H.N., Chen, R., Nishimura, R., Jennings, J.L., Link, A.J., and Zhou, Q.
(2003). Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II
transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol.
Cell 12, 971982.
Yoh, S.M., Schneider, M., Seifried, J., Soonthornvacharin, S., Akleh, R.E.,
Olivieri, K.C., De Jesus, P.D., Ruan, C., de Castro, E., Ruiz, P.A., et al.
(2015). PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate
Response to HIV-1. Cell 161, 12931305.
Zhang, G., Chan, B., Samarina, N., Abere, B., Weidner-Glunde, M., Buch, A.,
Pich, A., Brinkmann, M.M., and Schulz, T.F. (2016). Cytoplasmic isoforms of
Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune
DNA sensor cGAS. Proc. Natl. Acad. Sci. USA 113, E1034E1043.

Molecular Cell 67, 113, August 3, 2017 13

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Anti-HEXIM1 antibody - ChIP Grade (WB: 1/3000; IP: 2mg/ml; Abcam ab25388
IF: 1/500)
Anti- Cyclin T1 antibody - (WB: 1/3000 ; IP: 5mg/ml) Santa Cruz Biotechnology sc-10750
Anti-Cdk9 antibody - (WB: 1/500) Santa Cruz Biotechnology sc-13130
Anti-LARP7 antibody - (WB: 1/2000) Bethyl Laboratories A303-723A
Anti-KU70 antibody - (WB: 1/2000 ; IP: 2mg/ml ; IF: 1/500) Bethyl Laboratories A302-624A
Anti-KU80 antibody - (WB: 1/2000) Bethyl Laboratories A302-627A
Anti-DNA PKcs antibody - (WB: 1/1000) Abcam ab70250
Anti-SFPQ antibody - (WB: 1/1000 ; IP: 2mg/ml) Bethyl Laboratories A301-320A
Anti-SFPQ antibody - (WB: 1/1000 ; IF: 1/500) Sigma P2860
Anti-NONO antibody - (WB: 1/2000) Bethyl Laboratories A300-587A
Anti-PSPC1 antibody - (WB: 1/1000) Bethyl Laboratories A303-206A
Anti-RIF1 antibody - (WB:1/1000) Bethyl Laboratories A300-568A
Anti-NAK/TBK1 antibody (WB: 1/1000) Abcam ab109735
Anti-NAK/TBK1 (phospho S172) antibody - (WB: 1/1000) Abcam ab109272
Anti-IRF3 antibody - (WB: 1/1000) Abcam ab25950
Anti-PQBP1 antibody - (WB: 1/1000) Bethyl Laboratories A302-801A
Anti-STING antibody - (WB: 1/1000) Cell Signaling Technology D2P2F
Anti-MB21D1 (cGAS) antibody - (WB: 1/500 ; IP: 1mg/ml) Sigma-Aldrich HPA031700
Anti-Rig-I antibody - (WB: 1/1000) Cell Signaling Technology D33H10
Anti-Actin antibody - (WB: 1/5000) Sigma-Aldrich A4700
Anti-FLAG M2 antibody, Clone M2 - (WB: 1/1000) Sigma-Aldrich F1804
Anti-HA antibody, Clone 3F10 - (WB: 1/1000) Roche Cat#11867423001
Anti-Myc antibody - (WB: 1/2000; IP: 1mg/ml) Abcam ab9106
Anti-rabbit IgG, HRP-linked Antibody - (WB: 1/2000) Cell Signaling Technology 7074
Anti-mouse IgG, HRP-linked Antibody - (WB: 1/2000) Cell Signaling Technology 7076
Anti-Rat IgG, HRP-linked Antibody (WB: 1/2000) GE Healthcare NA935
Anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor-488 Invitrogen A11008
conjugate - (IF: 1/250)
Chemicals, Peptides, and Recombinant Proteins
3X FLAG Peptide Sigma-Aldrich F4799
HA Peptide Roche Cat#11666975001
rh IFN-alpha 2a ImmunoTools Cat#11343506
Critical Commercial Assays
EZview Red ANTI-FLAG M2 Affinity Gel Sigma-Aldrich F2426
HA-probe (F-7) Santa Cruz Biotechnology sc-7392
RNase Cocktail Enzyme Mix Ambion AM2286
Recombinant RNasin Ribonuclease Inhibitor Promega N2515
PhosSTOP Roche Cat# 04906837001
RNA-seq kits (see Extended Experimental procedures) N/A
ISD/LyoVec Invivogen tlrl-isdc
Poly(I:C) (HMW) / LyoVec Invivogen tlrl-piclv
20 30 -cGAMP Invivogen tlrl-cga23
(Continued on next page)

e1 Molecular Cell 67, 113.e1e5, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
SuperScript IV Reverse Transcriptase kit Invitrogen 18090050
QuantiTect SYBR Green PCR Kit QIAGEN 204143
Stellaris FISH probes Biosearch Technologies SMF-2248-5 ; SMF-2244-5
Deposited Data
Unprocessed image files used to prepare the figures in This study http://dx.doi.org/10.17632/968jfbz8xd.1
this manuscript
RNA-seq raw data This study SRA: SRR5340085, SRR5340084,
SRR5340083
Experimental Models: Cell Lines
HeLa S3 ATCC CCL-2.2
HeLa ATCC CCL-2
HUVEC/TERT2 ATCC CRL-4053
HEK293-rKSHV.219 Gift from Paivi M. Ojala N/A
(University of Helsinki)
Oligonucleotides
A full list of siRNA is presented in Table S1 MWG-Biotech N/A
Sigma-Aldrich
A full list of qPCR primers is presented in Table S2 MWG-Biotech N/A
Recombinant DNA
pAdRSV-Flag containing the HEXIM1 WT or mutants (Michels et al., 2004) and N/A
(ILAA ; 1-180 ; 181-359; 150-359) gift from Olivier Bensaude
(ENS, Paris)
pOZ-FLAG/HA-HEXIM1 WT IL2 R This study and (Nakatani N/A
and Ogryzko, 2003)
pCDNA4-MYC containing KSHV ORF52 Gift from Thomas Schulz N/A
Software and Algorithms
STAR software v. 2.4.0g1 (Dobin et al., 2013; Anders N/A
et al., 2015)
htseq-count software v. 0.6.1p1 (Anders et al., 2015) N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Monsef
Benkirane (monsef.benkirane@igh.cnrs.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lines
Adherent or suspension cells (HeLa, HeLa S3, 293 T) were grown in Dulbeccos modified Eagles medium (DMEM) supplemented
with 10% of fetal calf serum, antibiotics (100 units/ml penicillin G, 100mg/ml streptomycin), and ultraglutamine (0,2mM). All cell cul-
ture reagents were purchased from Lonza. HUVEC cells (Human umbilical vein endothelial cells) were purchased from ATCC and
cultured in EGM-2 medium supplied with growth factors (EGM-2 Bullet kit from Lonza). HEK293 cells infected with rKSHV.219
(293-rKSHV.219 cells) were kindly provided by Paivi OJALA and maintained in DMEM supplemented with 10% FBS, 1% peni-
cillin/streptomycin and 1mg/ml puromycin.

METHOD DETAILS

Plasmids
pAdRSV-Flag containing the HEXIM1 WT or mutants (ILAA ; 1-180 ; 181-359; 150-359) were a kind gift of Oliver Bensaude. The
HEXIM1 WT sequence was introduced into the pOZ-Flag/HA (F/H) IL2 R expression vector with Flag and HA tags at the N terminus
(Nakatani and Ogryzko, 2003). pCDNA4-MYC containing KSHV ORF52 was a kind gift of Thomas Schulz.

Molecular Cell 67, 113.e1e5, August 3, 2017 e2

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Virus production and cell line transduction

293T cells were transfected with a packaging mixture and the retroviral vector pOZ, using the calcium phosphate precipitation
method. For transfection, 5mg of the retroviral vector, 2.5mg of the MMLV packaging plasmid (gag/pol) and 2.5mg of the A-MLV
envelope plasmid were mixed with 125mL of CaCl2 (1M) and 500mL of HBS2X (sigma) in a final volume of 1mL. The mixture was incu-
bated for 1min at room temperature and then added dropwise to the cells. The medium was changed the following day and the viral-
containing supernatant was collected 48 hr after transfection, filtered through a 0.45 mm filter and subsequently used to infect cells.
HeLa or HeLa S3 cells were transduced with recombinant retroviruses expressing a bicistronic mRNA that encodes the tagged pro-
tein and a selection marker (IL-2 receptor subunit alpha). Transduced cells were purified by affinity cell sorting as previously
described (Nakatani and Ogryzko, 2003).

Purification of HEXIM1-associated complexes

Nuclear extracts were prepared using the Dignam protocol with slight modifications (Dignam et al., 1983). For the purification of
HEXIM1-associated complexes, 2x108 HeLa S3 cells stably expressing Flag HA tagged HEXIM1 and control HeLa S3 were harvested
by centrifugation, washed in cold PBS 1x and resuspended in 3 packed cell pellet volumes of hypotonic buffer (20 mM Tris-HCl
pH 7.4, 10 mM NaCl, and 1.5 mM MgCl2). The suspension was incubated on ice for 10 min and then cells were lysed using a Dounce
homogenizer fitted with a B pestle. The nuclei were pelleted by centrifugation and resuspended in one packed nuclear pellet volume
of a buffer containing 20 mM Tris-HCl pH 7.4, 0,02 M NaCl, 20% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2, 0,5mM phenylmethylsul-
fonyl fluoride (PMSF) and 1mM DTT. One packed nuclear pellet volume of a high salt buffer (containing 20 mM Tris-HCl pH 7.4,
1,2 M NaCl, 20% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2 0,5mM PMSF and 1mM DTT) was added dropwise to the suspension under
gently stirring with a magnetic bar. After stirring for 30 min to allow extraction of transcription factors, the suspension was centrifuged
at 25,000 g for 30 min at 4 C and the supernatant was dialyzed against 100 volumes of buffer BC150 (20 mM Tris-HCl pH 7.4, 150 mM
NaCl, 20% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2 and PMSF) for 4 hr. The dialysate (nuclear extract) was cleared by centrifugation at
25,000 g for 30 min at 4 C. Nuclear extracts were incubated overnight (at 4 C with rotation) with anti-FLAG M2 agarose beads (Sigma)
(1% v/v) equilibrated in BC150. Beads were washed 3 times with 10 mL buffer TETNG-150 (20 mM Tris-HCl pH 7.4, 150 mM NaCl,
10% glycerol, 0.5 mM EDTA, 5 mM MgCl2, 0.05% Triton X-100, 0.1% Tween; 10% glycerol, 1mM DTT and 0.5 mM PMSF) and bound
proteins were eluted with 4 bead volumes of B015 containing 2.5 mg/mL of FLAG peptide (Sigma) for 1 hr. The FLAG affinity purified
complexes were further immuno-purified by affinity chromatography using 20 mL of anti-HA conjugated agarose beads (Santa Cruz).
After incubation for 4 hr, HA beads were washed 4 times with 1mL of buffer TETNG-150 and eluted under native conditions using HA
peptide (Roche). Ten percent of the eluate was resolved on SDS-PAGE and Silver stained using a Silverquest kit (Invitrogen). The
remaining eluate was resolved on SDS-PAGE then stained with Coomassie-R250 and subsequently analyzed by mass spectrometry
at the Taplin Biological Mass Spectrometry facility (Harvard Medical School, Boston, MA).
For RNase treatment of HEXIM1 complexes, nuclear extract were incubated in the presence or absence of 1ml/ml RNase cocktail
(RNase A at 500U/mL; RNaseT1 at 20,000U/ML) for 10min at 37 C and subjected to a Flag- and a HA immunoprecipitation as above.

Glycerol Gradient Sedimentation Analysis and Reciprocal Immunoprecipitations

For density gradient sedimentation, 100 mL of FLAG-purified material was loaded onto 4.5 mL of a 10% to 40% glycerol gradient
(40%-0.5ml / 35%-0.5ml / 30%-0.5ml / 25%-1ml / 20%-1ml / 10%-1ml) in buffer (10 mM Tris [pH 8], 150 mM NaCl, 10mM kCl,
1.5 mM MgCl2, 0.5% Triton, 0.5mM EDTA, 1mM DTT, 0.5 mM PMSF) and centrifuged for 5h at 55,000 rpm in a Beckman
SW55Ti rotor. 200 mL fractions were collected from the top of the gradient. An equal volume of each fraction was analyzed by immu-
noblotting using specific antibodies.
For Immunodepletion of nuclear extract, nuclear extracts were subjected to two rounds (overnight and 3h) of incubation with 5 mg
of anti-Cyclin T1 or irrelevant IgG. The supernatants were then subjected to a Flag immunoprecipitation. For reciprocal immunopre-
cipitation, 50 mL of FLAG-purified HEXIM1 complex was diluted in 150 mL buffer TETN-150G and incubated with the indicated anti-
bodies under gentle agitation at 4 C for 4h. After extensive washing with TETN-150G the beads were boiled in loading buffer.

Reagents, transfection and siRNA

Transfection of pAdRSV-Flag containing the HEXIM wt or mutants (ILAA ; 1-180 ; 181-359; 150-359) or pCDNA4-MYC containing
KSHV ORF52 was performed using JETPEI (Polyplus) according to the manufacturers protocol. RNA interference was achieved us-
ing INTERFERin (Polyplus) according to the manufacturers protocol. HeLa cells were transfected twice with 20nM of siRNA. At day 3,
the cells were treated with 10mg/ml ISD/LyoVec (Invivogen) or 1mg/ml Poly(I:C)/LyoVec (Invivogen) or 100 UI/ml IFNa (ImmunoTools)
and harvested 6h later. 20 -30 cGAMP (10mg/ml; Invivogen) was transfected for 6h using lipofectamine2000 in a 1:1 ratio (Invitrogen).
IFNa, IFNb, and Mxa mRNA were quantified by RT-qPCR using specific probes. Normalization was performed using GAPDH-specific
probes.

RNaseq
Twenty nanogrammes of mRNA-enriched fraction was used to construct sequencing libraries using Illuminas TruSeq Stranded
mRNA Sample Prep Kit (Low throughput). In brief, the mRNA was fragmented into small pieces using divalent cations under
elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase,

e3 Molecular Cell 67, 113.e1e5, August 3, 2017

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Actinomycine D and random hexamer primers. The second strand cDNA was synthesized by replacing dTTP with dUTP. These cDNA
fragments then had a single A base added and subsequent ligation of the adaptor. The products were then purified and enriched
with 15 cycles of PCR. The final cDNA libraries were validated with a DNA 1000 Labchip on a Bioanalyzer (Agilent) and quantified with
a KAPA qPCR kit. For one sequencing lane of a flowcell V3, four libraries were pooled in equal proportions, denatured with NaOH and
diluted to 7.5 pM before clustering. Cluster formation, primer hybridization and single-read 50-cycle sequencing were performed on a
cBot and a HiSeq2000 (illumina, San Diego, CA) respectively.
Image analysis and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. Demulti-
plexing was performed using Illuminas sequencing analysis software (CASAVA 1.8.2). The quality of the data was assessed using
FastQC from the Babraham Institute and the Illumina software SAV (Sequence Analysis Viewer). Potential contaminants were inves-
tigated with the FastQ Screen software from the Babraham Institute. Single-ended 50 bp reads were mapped to the last release of
Human genome (GRCh38/hg38) with STAR (v. 2.4.0g1) (Anders et al., 2015; Dobin et al., 2013). Reads located in Ensembl annota-
tions of GRCh38 r77 were then counted with htseq-count (v. 0.6.1p1) (Anders et al., 2015) and normalized according sequencing
depth and gene length (RPKM). Then we use the ratio of RPKM between two complexes (HEXIM1/CycT1 or HEXIM1/Ku70) to
see specific associated RNA enrichment.

Immuno-FISH
NEAT1 RNA-IMMUNOFISH was performed using Stellaris FISH probes (human, SMF2248-5; SMF-2244-5) according to the manu-
facturers protocol. In brief, HeLa cells were grown on coverslips in a 6-well culture plate. Cells were fixed with 3.7% (vol./vol.) form-
aldehyde in 1X PBS for 10 min. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Primary
antibodies were incubated for 30 min at 37 C. The coverslips were washed three times with PBS for 10 min each. Secondary anti-
bodies were incubated for 30 min at 37 C. After washing, coverslips were fixed with 3.7% (vol./vol.) formaldehyde in PBS for 10 min.
The fixed cells were incubated in the dark with NEAT1 RNA probes overnight at 37 C. The next days, coverslips were washed exten-
sively and mounted with Vectashield-DAPI Mounting medium.
As nuclei are 3D objects, speckle colocalization experiments were performed in 3D.
Images were acquired with a Carl Zeiss LSM780 confocal microscope. 3D stacks of images were acquired using a PL-APO 63x 1.4
lens. Voxel size, slightly undersampled compared to Shannon/Nyquist criterion, was optimized to allow the best combination
possible of sample size (number of analyzed nuclei) and accurate nuclei and spot detection. Tilescans were used, rather than series
of single fields of view stacks, to minimize rejection of nuclei at the edges of the final stack.
Confocal 3D stacks were analyzed using Imaris software (Oxford Instruments). First, nuclei were segmented using an automated
threshold (based on the intensity distribution histogram). Identified nuclei populations were filtered to remove large nuclei aggregates
(upper nuclei volume threshold) and fragments (lower threshold and manual removing of the remaining fragments of nuclei at the
edges of the stack). A visual segmentation result inspection was performed and remaining, rare, fused nuclei were manually sepa-
rated in 3D.
Next, speckles were identified in both channels using the Imaris spot tool. The seed spot size used was 0.3um. For the sake of
objective speckle center identification, automatic thresholds were used to filter raw spot quality. However, in a very few instances
of poor signal to noise ratio, a manual correction of the quality threshold was performed. Spot colocalization was quantified in
3D using an adaptation of the approach developed by Lachmanovich and colleagues. Spots were considered as colocalized if
distant by less than the spatial resolution of the system. As lateral and axial resolution values are different, an average value of
0.25um was used. The Colocalize spot Xtension command of Imaris was used for this purpose. Then, identified spots (colocalized
and non colocalized spots in both channels) were further reassigned to their respective nuclei using the split into surface Xtension
command.
Speckle total intensity was measured using the same tools (nuclei segmentation, spot detection, per nucleus spots dispatch),
except that different sizes of spots were allowed using an objective, automatic intensity threshold calculation. The total integrated
values were used for comparisons.

Whole cell extract preparation and IPs

All steps were performed on ice or in a cold room. Following harvest, cells were washed with cold 1X PBS and then resuspended in
5 cell pellet volumes of TETN-150 buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 0.5 mM EDTA, 5 mM MgCl2, 0.05%
Triton X-100, 0.1% Tween; 1mM DTT and 0.5 mM PMSF) and incubated for 30 min on wheel at 4 C. After a 30 min centrifugation at
14,000 rpm the extract was transferred to a fresh tube. Protein were quantified by Bradford assay. Antibodies and protein A or G
beads (for antibodies produced in rabbits or mice respectively) were added for overnight incubation. All FLAG-IPs were performed
using anti-FLAG-M2-agarose (Sigma). After three washes in TETNG-150, the beads were incubated with 4 volumes of FLAG peptide
solution (Sigma) that had been diluted in TETNG-150 at 2.5 mg/ml for one hour at 4 C. Samples were resolved by SDS-PAGE and
proteins transferred onto a nitrocellulose membrane.

RNA extraction and RT-qPCR

RNA was extracted using Trizol (Invitrogen) or RNeasy plus Mini Kit (QIAGEN) according to the manufacturers protocols. Reverse
transcribed products were obtained using hexamer primers and according to the manufacturers instruction (SuperScript III or IV

Molecular Cell 67, 113.e1e5, August 3, 2017 e4

 Please cite this article in press as: Morchikh et al., HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-
Mediated Innate Immune Response, Molecular Cell (2017), http://dx.doi.org/10.1016/j.molcel.2017.06.020

Reverse Transcriptase kit Invitrogen). The qPCR experiments were performed using a standard protocol (QuantiTect SYBR Green
PCR Kit; QIAGEN).

KSHV viral production

rKSHV.219 latently infected HEK293 cells were plated and used to purify recombinant virus. When the monolayer reached 60%
confluence, cells were reactivated by incubating them in DMEM medium containing 1 mM sodium butyrate and 20 ng/mL PMA
(phorbol 12-myristat 13-acetate) for 24 hr, and for two more days with medium containing sodium butyrate only. The supernatant
was collected, filtered through a 0.45 mM filter, and virions were then pelleted by ultracentrifugation on a 20% sucrose cushion at
100,000 3 g for 1 h30 with a Beckman SW28 rotor. Pellets were dissolved in 0.2% of the original volume of EBM2 and stored
at 80 C. KSHV DNA was extracted from the purified virus, and copy numbers were quantified by qPCR using primers amplifying
KSHV ORF50 and LANA regions.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data in Figures 4 and 6 and S4 are represented as the mean standard deviation of the mean (SD), as stated in the figure legends.
Data in Figures S1, S3, and S6 show one representative experiment.

DATA AND SOFTWARE AVAILABILITY

The accession numbers for the RNA sequencing data reported in this paper are SRA: SRR5340085, SRR5340084, and SRR5340083.
The unprocessed image files are in Mendeley Data and are available at: http://dx.doi.org/10.17632/968jfbz8xd.1

e5 Molecular Cell 67, 113.e1e5, August 3, 2017