Volume 67, Issue 3, Pages 387-399.e5 (August 2017) HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response  Mehdi Morchikh, Alexandra Cribier, Raoul Raffel, Sonia Amraoui, Julien Cau, Dany Severac, Emeric Dubois, Olivier Schwartz, Yamina Bennasser, Monsef Benkirane  Molecular Cell  Volume 67, Issue 3, Pages 387-399.e5 (August 2017) DOI: 10.1016/j.molcel.2017.06.020 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 immunoprecipitation (CycT1 IP). Input as well as flow-through (FT) from CycT1 IP were probed with the indicated antibodies (lane 1–8). 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 13–16). 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. Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 4–6); a non-targeting siRNA (siSCR, lane3) was used as a control. Whole-cell extracts were then prepared, treated with RNase or not (lanes 1–3), 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 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 The HDP-RNP Is Involved in the cGAS-STING-IRF3-Dependent Innate Immune Response to ISD (A) Upper panel: HeLa cells were transfected with siRNA targeting HEXIM1, Ku70, NEAT1, SFPQ, or a non-targeting siRNA (SCR). Transfected cells were mock-treated (NS) or treated for 6 hr with 10 μg/mL ISD or 1 μg/mL Poly(I:C) or 100 IU/mL IFNα. The levels of induction of IFNα, IFNβ, and MXA mRNAs were measured by qRT-PCR. The graph shows mean ± SD (n = 3) of the fold induction relative to the control condition (siRNA SCR NS). Lower panel: whole-cell extracts were analyzed by western blotting using the indicated antibodies. (B) Upper panel: experiment performed as in (A) except that siRNA-transfected cells were mock-treated (NS) or treated for 6 hr with 10 μg/mL ISD or 2′-3′cGAMP. Induction of IFNβ was measured by qRT-PCR. The graph shows mean ± SD (n = 2) of the fold induction relative to the control condition (siSCR NS). Lower panel: whole-cell extracts from the experiment performed in the upper panel were analyzed by western blotting using the indicated antibodies. See also Figure S4. Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Regulation of the HDP-RNP Complex in Response to DNA Stimulation (A) Western blots of whole-cell extracts prepared from HeLa eHEXIM1-overexpressing cells that were mock-treated or treated for 6 hr with interferon stimulatory DNA (10 μg/mL) (lanes 1–3). These extracts were subjected to FLAG IP (lanes 4–6). The bulk of the FLAG-immunoprecipitated complexes (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 7–13). The presence of HDP-RNP complex subunits in FLAG-IP and reIPs was assessed by western blot (WB) using the indicated antibodies. (B) Whole-cell extracts prepared from HeLa cells transfected with siRNA targeting HEXIM1 that were mock-treated or treated for 6 hr with ISD (10 μg/mL) and subjected to endogenous cGas IP using a specific antibody. The presence of HDP-RNP complex subunits in the whole-cell extracts and IPs was assessed by WB using the indicated antibodies. See also Figure S5. Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

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 IFNβ 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 μg/mL) were subjected to IP of endogenous HEXIM1 (lanes 5–9) or MYC IP using specific antibodies. Input and bound complexes were analyzed by WB using the indicated antibodies. See also Figure S6. Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Proposed Model for the HDP-RNP Complex Interaction with the cGAS-STING-IRF3 Innate Immune Pathway and Its Regulation upon DNA Stimulation Molecular Cell 2017 67, 387-399.e5DOI: (10.1016/j.molcel.2017.06.020) Copyright © 2017 Elsevier Inc. Terms and Conditions