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. 2023 Aug;299(8):105071.
doi: 10.1016/j.jbc.2023.105071. Epub 2023 Jul 18.

The disordered C terminus of ALKBH5 promotes phase separation and paraspeckles assembly

Xiaoyang Qin  1 , Yan Long  1 , Xue Bai  2 , Lei Cao  1 , Han Yan  1 , Kai Zhang  2 , Bo Wang  3 , Xudong Wu  4
Affiliations

The disordered C terminus of ALKBH5 promotes phase separation and paraspeckles assembly

Xiaoyang Qin et al. J Biol Chem. 2023 Aug.

Abstract

Paraspeckles (PS) are nuclear structures scaffolded by the long noncoding RNA NEAT1 and protein components such as NONO and SFPQ. We previously found that the upregulation of RNA N6-methyl-adenosine (m6A) demethylase ALKBH5 facilitates hypoxia-induced paraspeckle assembly through erasing m6A marks on NEAT1, thus stabilizing it. However, it remains unclear how these processes are spatiotemporally coordinated. Here we discover that ALKBH5 specifically binds to proteins in PS and forms phase-separated droplets that are incorporated into PS through its C-terminal intrinsically disordered region (cIDR). Upon exposure to hypoxia, rapid ALKBH5 condensation in PS induces m6A demethylation of NEAT1, which further facilitates PS formation before the upregulation of ALKBH5 expression. In cells expressing ALKBH5 lacking cIDR, PS fail to be formed in response to hypoxia, accompanied with insufficient m6A demethylation of NEAT1 and its destabilization. We also demonstrate that ALKBH5-cIDR is indispensable for hypoxia-induced effects such as cancer cell invasion. Therefore, our study has identified the role of ALKBH5 in phase separation as the molecular basis of the positive feedback loop for PS formation between ALKBH5 incorporation into PS and NEAT1 stabilization.

Keywords: ALKBH5; NEAT1; m(6)A; paraspeckle; phase separation.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
ALKBH5 interacts with proteins in paraspeckles.A, identification of ALKBH5-interacting protein by mass spectrometry. The list of proteins specifically identified in HA affinity purifications followed by mass spectrometry analysis in HA-ALKBH5-overexpressed 293T cells. The number of unique peptides, coverage of the whole protein, and the molecular weight (MW, kDa) are shown. B and C, immunoprecipitation (IP) using the anti-GFP affinity gel, respectively, in EGFP-ALKBH5 (thereafter short as EGFP-A5) (B) or EGFP-SFPQ (C) overexpressed U87 cells, followed by Western blot analysis with designated antibodies. Protein A/G beads served as a negative control (ctrl). D and E, SFPQ immunostaining was performed in EGFP-ALKBH5-overexpressed U87 cells. Representative images are shown (D). The scale bars represent 2 μm. The fluorescence intensity curves are shown (E). F, NEAT1 RNA levels in U87 cells transduced with shRNA construct targeting NEAT1 (shNEAT1) or scramble shRNA (shScr) were determined by real-time quantitative PCR. Two-tailed unpaired Student’s t test. ∗∗, p < 0.01, n = 3. G, left panel: GFP pull-down to compare the interaction between ALKBH5 and SFPQ in NEAT1-deficient or control U87 cells overexpressing EGFP-ALKBH5. Protein A/G beads served as a negative control (ctrl). Right panel: statistical analysis to compare the relative SFPQ enrichment in the two groups (n = 3). H, immunofluorescence of SFPQ in NEAT1-deficient or control U87 cells overexpressing EGFP-ALKBH5. The scale bars represent 2 μm.
Figure 2
Figure 2
ALKBH5 phase separates through its C-terminal intrinsically disordered region (IDR) in vitro.A, top panel: schematic diagram of the ALKBH5 structure. Bottom panel: prediction of ALKBH5 IDRs using PONDR. Disordered amino acids are highlighted in black. B, representative confocal images of droplet formation experiments with EGFP-ALKBH5 protein concentration gradients. EGFP alone is included as a control. All of the assays were performed in the presence of 150 mM NaCl, and 10% PEG-6000 was used as a crowding agent. The scale bars represent 2 μm. C and D, left panel: representative confocal images of EGFP-ALKBH5 droplets treated with different concentrations of NaCl (C) or 1,6-hexanediol (D). All experiments were performed in the presence of 200 μM EGFP-ALKBH5 and 150 mM NaCl, and 10% PEG-6000 was used as a crowding agent. The scale bars represent 2 μm. Right panel: statistical analysis to compare droplet numbers at each condition (n = 3). E, fluorescence recovery after photobleaching of EGFP-ALKBH5 droplets. Representative images are shown in the top panel. The protein concentration was 800 μM. Droplet formation assays were performed with protein concentration 800 μM in the presence of 150 mM NaCl and 10% PEG-6000. The scale bars represent 1 μm. Quantitative analysis of the average recovery of fluorescence of EGFP-ALKBH5 is shown in the bottom panel. Data are presented as mean ± SEM (n = 5). F, top panel: schematic diagram of the full length or truncates of EGFP-ALKBH5 proteins. Bottom panel: representative confocal images of a concentration series of droplet formation assays testing droplet formation of the full length or truncates of EGFP-ALKBH5 proteins. EGFP alone is included as a control. All of the assays were performed in the presence of 150 mM NaCl and 10% PEG-6000. The scale bars represent 2 μm.
Figure 3
Figure 3
ALKBH5 phase separates and incorporates into paraspeckles through its C-terminal intrinsically disordered region (cIDR).A, representative confocal images of U87 cells expressing the full length or truncates of EGFP-ALKBH5 proteins. The scale bars represent 2 μm. B, the paraspeckle numbers were counted for 20 corresponding U87 cells. Two-tailed unpaired Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001. C, SFPQ immunostaining was performed in U87 cells expressing EGFP-A5WT or EGFP-A5dcIDR. The scale bars represent 2 μm.
Figure 4
Figure 4
Rapid ALKBH5 condensation is coupled with m6A demethylation of NEAT1 and paraspeckles assembly in response to hypoxia.A, ALKBH5 immunostaining was performed in NEAT1-deficient or control U87 cells after 1 h of hypoxia treatment. Representative images are shown. The scale bars represent 2 μm. B, MeRIP (m6A-IP)-qPCR analysis of the NEAT1 m6A peak region in U87 cells at different time points of hypoxia treatment. Two-tailed unpaired Student’s t test. ∗p < 0.05, n = 3. C, real-time quantitative PCR analysis of the RNA expression levels of NEAT1-2 in U87 cells at different time points of hypoxia treatment. Two-tailed unpaired Student’s t test. ∗, p < 0.05, ∗∗, p < 0.01, n = 3. D, SFPQ immunostaining was performed in U87 cells after 1 h of hypoxia treatment. Representative images are shown. The scale bars represent 2 μm.
Figure 5
Figure 5
ALKBH5-cIDR is required for hypoxia-induced m6A demethylation of NEAT1 and paraspeckles assembly.A, MeRIP (m6A-IP)-qPCR analysis of the NEAT1 m6A peak region in ALKBH5-deficient U87 cells rescued by A5-WT or dcIDR mutant under hypoxic conditions. Two-tailed unpaired Student’s t test. ∗p < 0.05, n = 3. B, real-time quantitative PCR analysis of RNA expression levels of NEAT1-2 in corresponding U87 cells under hypoxic conditions. Two-tailed unpaired Student’s t test, ∗∗, p < 0.01, n = 3. C, SFPQ immunostaining was performed in corresponding U87 cells under hypoxic conditions. Representative images are shown. The scale bars represent 2 μm. D, the number of paraspeckles was counted and analyzed in 20 corresponding U87 cells. Two-tailed unpaired Student’s t test, ∗∗∗p < 0.001.
Figure 6
Figure 6
ALKBH5-cIDR is required for hypoxia-induced effects.A and B, transwell assays (A) were performed to measure cIDR's ability to promote invasion in ALKBH5-deficient U87 cells under hypoxia. The scale bars represent 100 μm. The statistical chart of the invasion cells is given in (B). Two-tailed unpaired Student’s t test, ∗∗∗p < 0.001, n = 3. C and D, VEGFA RNA levels (C) and LDHA RNA levels (D) in corresponding U87 cells were determined by real-time quantitative PCR analysis. Two-tailed unpaired Student’s t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3. cIDR, C-terminal intrinsically disordered region.
Figure 7
Figure 7
A working model for ALKBH5-cIDR in phase separation and paraspeckle assembly. In response to stress such as hypoxia, ALKBH5 undergoes condensation through its cIDR and facilitates paraspeckles assembly, in which NEAT1 m6A is demethylated and stabilized and therefore fosters a positive back. If the cIDR of ALKBH5 is truncated, it is inefficient to target NEAT1 for m6A demethylation, leading to NEAT1 instability and compromised paraspeckles assembly. cIDR, C-terminal intrinsically disordered region; nIDR, N-terminal intrinsically disordered region.
Supporting Figure S1
Supporting Figure S1
Supporting Figure S2
Supporting Figure S2
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