Figure 3. NEAT1 interacts with PGK1, PGAM1 and ENO1.
(A) Lysates from MCF7 cells were subjected to NEAT1 RNA immunoprecipitation (RIP) with IgG and anti-PGK1, –PGAM1, –ENO1 or –GAPDH.
(B) Lysates from MCF7 cells were subjected to CLIP-qPCR for NEAT1_1 segments, as shown in top diagram, with IgG and anti-PGK1, –PGAM1 or –ENO1. A secondary structure model of NEAT1 (Lin et al., 2018) in bottom right. The four structural domains are highlighted with different colors, and the NEAT1_1 sequence motifs that are recognized by PGK1, PGAM1 and ENO1 are drawn as dashed lines.
(C, F) Generation of MCF7 cell lines lacking the NEAT1 sequence motifs that are recognized by PGAM1 and ENO1 (Δ1033~2115 bp; Δ1~2.1k) (C) or PGK1 (Δ2116~2805 bp; Δ2.1~2.8k) (F) using CRISPR/Cas9 technology with a series of sgRNAs (left). Lysates from wild-type (WT) and Δ1~2.1k or Δ2.1~2.8k cells were subjected to NEAT1 RIP with IgG and anti-PGK1, –PGAM1 or –ENO1 (right). n = 3~4.
(D, G) Glucose consumption (left) and lactate production (right) by WT and Δ1~2.1k (D) or Δ2.1~2.8k (G) cells were determined by enzymatic assays. n = 3.
(E, H) Intracellular concentrations (nmol) of glycolytic metabolites, as indicated, per 1 mg of protein of WT and Δ1~2.1k (E) or Δ2.1~2.8k (H) cells were measured by CE-MS based and colorimetric assays. n = 3~4.
(I, L, O) Lysates from MCF7 cells expressing PGK1 (I), PGAM1 (L) or ENO1 (O) shRNA together with exogenous WT or K/R-to-A mutants, as indicated, PGK1, PGAM1 or ENO1 from an ORF transcript lacking the 3’-UTR sequences targeted by shRNA were subjected to RIP-qPCR for NEAT1 with IgG and anti-PGK1, –PGAM1 or –ENO1 (left). Structure-based prediction of NEAT1 and PGK1, PGAM1 or ENO1 interaction (right). Basic residues are displayed in blue and acidic residues are shown in red. The PGK1, PGAM1 and ENO1 protein structures were visualized with Swiss-PdbViewer (PDBID:3C39, PDBID:4GPI and PDBID:3B97, respectively).
(J, M, P) Glucose consumption (μmol/106 cells) of (top) and lactate production (μM) (bottom) by MCF7 cells expressing PGK1 (J), PGAM1 (M) or ENO1 (P) shRNA together with exogenous WT or K/R-to-A mutants, as indicated, PGK1, PGAM1 or ENO1 were determined by enzymatic assays. n = 3~4.
(K, N, Q) Intracellular concentrations (nmol) of glycolytic metabolites, as indicated, per 1 mg of protein of MCF7 cells expressing PGK1 (K), PGAM1 (N) or ENO1 (Q) shRNA together with exogenous WT or K/R-to-A mutants, as indicated, PGK1, PGAM1 or ENO1 were determined by colorimetric assays. n = 3.
Error bars represent +/− SD. p value was determined by Student’s t test (n.s., non-significant; *p<0.05; **p<0.01; ***p<0.001). See also Figure S3.