Research ArticleCELL BIOLOGY

LncRNA GIRGL drives CAPRIN1-mediated phase separation to suppress glutaminase-1 translation under glutamine deprivation

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Science Advances  24 Mar 2021:
Vol. 7, no. 13, eabe5708
DOI: 10.1126/sciadv.abe5708
  • Fig. 1 GIRGL negatively regulates cellular glutamine metabolism.

    (A) Expression of GIRGL (qPCR, top) and GLS1 (Western blot, bottom) in HCT116 cells transduced with control (PCDH) or GIRGL-containing (PCDH-GIRGL) vectors. Relative GLS1 levels were determined throughout by densitometry against the β-actin loading control. (B) Expression of GLS1 following shRNA-mediated silencing of GIRGL alone or in combination with an shRNA-resistant GIRGL (GIRGL-R) construct. (C and D) GLS1 expression in HT29 cells following silencing of GIRGL using two independent shRNAs (sh-1 and sh-2) (C) or following GIRGL overexpression (D). (E) Intracellular glutaminase activity in HCT116 cells transduced with shCtrl or shRNAs targeting GIRGL after culture with or without 2 mM glutamine for 24 hours. (F to H) Cellular α-KG levels (F), ROS levels (G), and GSH levels (H) in HCT116 cells transduced with shCtrl or independent shRNAs targeting GIRGL (sh-1 and sh-2). (I) Protein synthesis in HCT116 cells transduced with shCtrl or shRNAs targeting GIRGL cultured with or without 2 mM glutamine for 24 hours. FITC, fluorescein isothiocyanate. (J) Intracellular glutamine, glutamate, aspartate, and asparagine levels in HCT116 cells as per (I). (K) RNA synthesis in HCT116 cells as per (I). (L) DNA synthesis in HCT116 cells as per (I). Epifluorescence microscopy compares de novo DNA synthesis (EdU; red) versus total (green). Representative fields (left) and quantitation (right). (A) to (F) and (H) to (L) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test. (G) represents three independent experiments (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 2 Glutamine deprivation drives c-Jun–mediated transactivation of GIRGL.

    (A) Exon-specific qPCR measuring GIRGL in HCT116 cells cultured without glutamine for 0 to 48 hours. (B) Protein expression of ESX1, CLOCK, ATF4, ATF7, CDX1, DUX1, c-Jun, P-c-Jun, HIF1α, and c-Myc in HCT116 cells in response to glutamine deprivation. β-Actin served as loading control throughout. (C) GIRGL (qPCR, top) and c-Myc/c-Jun protein levels (bottom) in HCT116 cells after shRNA-mediated knockdown of c-Myc (left) or c-Jun (right). (D) Levels of GIRGL (top) and ectopic c-Jun protein (anti-Flag, bottom) in HCT116 cells transduced with control (pCMV-3×Flag) or Flag–c-Jun (pCMV-3×Flag–c-Jun). (E) ChIP assays against control immunoglobulin G (IgG) or anti–p-c-Jun (Ser73) in HCT116 cells targeting the c-Jun consensus binding site within the GIRGL proximal promoter (top). Semiquantitative PCR (bottom left) or qPCR-based detection (bottom right) of GIRGL and negative (GAPDH) and positive (MMP1) controls. (F) pGL3-based reporter plasmids were constructed around the c-Jun binding sequence (1238 to 1250) in the GIRGL proximal promoter (E), and dual-luciferase assays were performed in HCT116 cells using pGL3 control or GIRGL reporters containing either wild-type or mutant c-Jun binding sequences (top) with or without ectopic Flag–c-Jun (bottom). (A) and (C) to (F) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test. (B) represents three independent experiments (*P < 0.05; **P < 0.01).

  • Fig. 3 HuR negatively regulates GIRGL expression under glutamine deprivation.

    (A) GIRGL (qPCR; top) or c-Jun protein levels (bottom) in HCT116 cells after shRNA-mediated knockdown of c-Jun in combination with glutamine depletion. (B) Half-life time of GIRGL in HCT116 cells cultured with or without glutamine for 48 hours before addition of actinomycin (5 μg/ml) for 0 to 9 hours. (C) RNA pull-down assays were conducted in HCT116 cells against biotin-labeled sense/antisense GIRGL probes, with HuR and β-actin proteins detected by Western blotting. (D) RIP assays with IgG or HuR antibodies were performed in HCT116 cells, and the levels of GIRGL (top) and recovered target proteins (bottom) were measured by qPCR and Western blot, respectively. (E) GIRGL (qPCR, top) and HuR protein level (bottom) measurements in HCT116 cells after silencing HuR using two independent shRNAs. (F) GIRGL levels were determined as per (D) following transduction of control (pCMV-3×Flag) or Flag-HuR (pCMV-3×Flag-HuR) (top). Ectopic HuR was revealed with anti-Flag antibodies (bottom). (G) Half-life time of GIRGL in HCT116 cells transduced with shCtrl or shHuR before addition of actinomycin D (5 μg/ml) for 0 to 9 hours. (H) HuR protein measurements in HCT116 cells following glutamine deprivation for 0 to 48 hours. (I) RNA pull-down assays conducted as per (C) in HCT116 cells cultured with or without glutamine. (A), (B), and (D) to (G) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test. (C), (H), and (I) represent three independent experiments (*P < 0.05; **P < 0.01).

  • Fig. 4 GIRGL suppresses the translation of GLS1.

    (A) GLS1 protein levels in HCT116 cells transduced with shCtrl or independent shRNAs against GIRGL (shGIRGL-1, shGIRGL-2) after culture without glutamine (0 to 48 hours). β-Actin served as loading control. (B) GLS1, HuR, or β-actin proteins captured in RNA pull-down assays using HCT116 cell lysates against biotin-labeled sense (Ctrl) or antisense GIRGL probes. (C and D) GLS1 mRNA levels in HCT116 cells after transduction with shCtrl or independent shRNAs against GIRGL (C) or cultured without glutamine for 0 to 48 hours (D). (E) GLS1 protein levels in HCT116 cells transduced with shCtrl or shGIRGL after treatment with 50 μM cycloheximide (CHX) for 0 to 12 hours (left). Densitometric analyses (right). (F) CDH1 protein expression after silencing of GIRGL in HCT116 cells using two independent shRNAs. (G) GLS1 protein levels in HCT116 cells treated with or without 20 μM MG132 for 6 hours following culture without glutamine for 0 to 48 hours (top). Densitometric analyses (bottom). (H) Velocity sedimentation analysis of HCT116 cells treated with or without glutamine for 48 hours. Continuous OD254nm measurements show resolution of 60S ribosomes, 80S monosome complex, and polysomes (left). The abundance of GLS1 mRNA relative to actin (right). (I) Velocity sedimentation as per (H) in HCT116 cells transduced with either shCtrl or shGIRGL-1. (J) Velocity sedimentation as per (H) in HCT116 cells transduced with either PCDH or PCDH-GIRGL. (A), (B), and (E) to (G) represent three independent experiments. (C), (D), and (H) to (J) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test.

  • Fig. 5 GIRGL cooperates with CAPRIN1 to suppress GLS1 translation.

    (A and B) GIRGL levels recovered using biotin-labeled sense/antisense GIRGL probes in RNA pull-down assays against HCT116 cells (A). SDS–polyacrylamide gel electrophoresis (left) and MS identified protein IDs (right) (B). (C) Putative GIRGL-binding proteins from (B) evaluated using Western blotting. (D) RIP assays performed against HCT116 cells using IgG, G3BP1, or CAPRIN1 antibodies. Western blot (top) with GIRGL levels (bottom). (E) GLS1 protein levels in HCT116 cells following transduction of control (Psin-3×Flag) or CAPRIN1 (Psin-3×Flag-CAPRIN1) vectors. Ectopic CAPRIN1 revealed using anti-Flag. (F) GLS1 protein levels in HCT116 cells following transduction of shCtrl, shCAPRIN1, shCtrl plus PCDH, and shCAPRIN1 plus PCDH-GIRGL. (G) Velocity sedimentation analysis of HCT116 cells transduced with shCtrl or shCAPRIN1. Polysome profiles (left) and abundance of GLS1 mRNA (right). (A), (D), and (G) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test. (C), (E), and (F) represent three independent experiments (**P < 0.01; ***P < 0.001).

  • Fig. 6 GIRGL promotes association between CAPRIN1 and GLS1 mRNA.

    (A) GLS1 mRNA (top) and CAPRIN1 protein levels (bottom) recovered with biotin-labeled sense (Ctrl) or antisense GLS1 probes in RNA pull-down assays against HCT116 cells. (B) GLS1 mRNA levels measured by qPCR in IgG and CAPRIN1 antibody RIP assays performed against HCT116 cell lysates. (C) Reciprocal RNA pull-down assays performed with sense/antisense GIRGL probes (left) or GLS1 probes (right) against HCT116 cells with recovery of GLS1 mRNA or GIRGL measured by qPCR. (D) GLS1 mRNA levels in RNA pull-down assays against GIRGL performed as per (C) after transducing cells with shCtrl or shCAPRIN1. (E) GLS1 mRNA levels in RIP assays conducted as per (B) in cells transduced with either shCtrl or shGIRGL. (F) GIRGL and GLS1 mRNA (top) and CAPRIN1 protein levels (bottom) recovered using antisense GIRGL probes in RNA pull-down assays using cells cultured with or without glutamine for 48 hours. (A) to (F) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test (*P < 0.05; **P < 0.01).

  • Fig. 7 GIRGL promotes CAPRIN1 dimerization, phase separation, and SG formation.

    (A) CAPRIN1 protein expression in HCT116 cells transduced with shCtrl or two independent shRNAs targeting GIRGL. β-Actin loading control was used throughout. (B) HCT116 cells as per (A) were treated with disuccinimidyl suberate (DSS) or dimethyl sulfoxide (DMSO) before analysis of CAPRIN1 homodimerization. (C) CAPRIN1 homodimerization as per (B) in HCT116 cells transduced with PCDH or PCDH-GIRGL. (D) CAPRIN1 homodimerization in GIRGL RNA pull-down samples from DMSO- or DSS-treated HCT116 cells. (E) Western blotting against Flag-CAPRIN1, HA-CAPRIN1 (left), and GIRGL qPCR (right) in two-step IPs conducted in HCT116 cells transfected with Flag-vector/HA-CAPRIN1 or Flag-CAPRIN1/HA-CAPRIN1. (F) Confocal images showing CAPRIN1 (green), G3BP1 (red), and DAPI (blue) staining in HCT116 cells transduced with PCDH or PCDH-GIRGL (scale bar, 10 μm). (G) Confocal images of GLS1 mRNA (FISH, green), GIRGL (FISH, yellow), and CAPRIN1 (IF, red) in HCT116 cells cultured with or without 2 mM glutamine for 48 hours (scale bar, 10 μm). (H) Recombinant Alexa Fluor 555–labeled Flag-CAPRIN1 (20 μM) was incubated with either GLS1 mRNA 3′UTR-1 or 5′UTR (0.2 μM) at room temperature. Epifluorescence images were collected to observe liquid-liquid phase separation droplets (left) (scale bar, 30 μm), and the normalized variance of droplets was measured as the index of dispersion (σ2/μ) (right). (I) Epifluorescence images were collected as per (H) after incubating Flag-CAPRIN1 (20 μM) with Alexa Fluor 546–labeled GLS1 mRNA 3′UTR-1 (0.2 μM) and Alexa Fluor 488–labeled sense-GIRGL or antisense-GIRGL (0.2 μM) at room temperature (left) (scale bar, 30 μm), and the normalized variance of droplets was measured as the index of dispersion (σ2/μ) (right). (A) to (D), (F), and (G) represent three independent experiments. (E), (H), and (I) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 8 Biological implications of GIRGL in tumorigenesis.

    (A to C) HCT116 cells were transduced with shCtrl or with one of two independent shRNAs targeting GIRGL (upper) and PCDH or PCDH-GIRGL (bottom) before culturing in 2 mM (A), 0.4 mM (B), and 0 mM (C) glutamine. Cell numbers were counted over consecutive days to assess proliferation. (D and E) Silencing of GIRGL promotes, whereas overexpression of GIRGL inhibits, the clonogenic potential of HCT116 cells. (F and G) HCT116 cells (2 × 106) transduced with shCtrl or shGIRGL-1 (F) or PCDH or PCDH-GIRGL (G) were inoculated into opposite flanks of nu/nu mice. Tumor sizes were measured at the indicated time points (left) and excised and weighed after 4 weeks (right) after mice were humanely culled. (H) Relative GIRGL expression among 30 pairs of matched colorectal cancer and adjacent normal tissues determined by qPCR. (A) to (C), (F), and (G) are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test. (D) and (E) represent three independent experiments (*P < 0.05; **P < 0.01).

  • Fig. 9 Working model for induction of GIRGL expression and down-regulation of GLS1 under glutamine deprivation conditions.

Supplementary Materials

  • Supplementary Materials

    LncRNA GIRGL drives CAPRIN1-mediated phase separation to suppress glutaminase-1 translation under glutamine deprivation

    Ruijie Wang, Leixi Cao, Rick Francis Thorne, Xu Dong Zhang, Jinming Li, Fengmin Shao, Lirong Zhang, Mian Wu

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