PMC

DJ-1 interacts with RNA in vivo. (a) IP for DJ-1 from whole brain lysates from knockout (lanes 1–3) or WT (lanes 4–6) mice. DJ-1 protein is absent from the knockout mice. (b) Validation by qRT-PCR after IP of DJ-1 from WT (closed bars) or knockout (open bars) mice. Error bars indicate the SEM (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 using t tests to compare genotypes.

The oxidation state of DJ-1 alters RNA interaction. (a) DJ-1 complexes were isolated from untreated (lane 2) or PQ-treated (lane 3) cells stably transfected with V5–6His-tagged WT DJ-1, or empty vector (lane 1) as a control, using NiNTA beads, then blotted for DJ-1. Arrows show V5–6His-tagged DJ-1, and arrowheads show endogenous DJ-1. (b and c) Associated RNA was analyzed by qRT-PCR (bars show mean signal, error bars indicate the SEM, n = 3 independent PQ treatments and pull downs per construct). PQ treatment decreases the binding of DJ-1 to the RNA for GPx4 and MAPK8IP1. Differences in qRT-PCR were analyzed by one-way ANOVA with Bonferroni's post hoc tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (d) PQ increases the protein levels of the GPx4 5′ UTR GFP construct in cells containing DJ-1. M17 cells were transfected with a reporter construct containing the 5′ UTR of GPx4 (see ) placed 5′ to EGFP and were either untreated (Control) or exposed to PQ. Cells were blotted for GFP or β-actin as a loading control. Quantification of n = 4 experiments is shown in the bar graph. *, P < 0.05 by t test.

Endogenous DJ-1 binds RNA. (a) DJ-1 was isolated by IP from M17 cells. (b) Associated RNA was isolated, amplified, and hybridized to arrays. Cluster analysis of replicate samples shows a strict separation of DJ-1 and control IPs. (c) Using two control (lanes 1 and 2) and three DJ-1 (lanes 3–5) IP samples, we performed RT-PCR for selenoproteins, mitochondrial transcripts, and components of the PTEN/Akt pathway; gene symbols are listed on the right of each gel. (d) Living M17 cells were subjected to UV cross-linking and immunoprecipitated with a nonspecific IgG (lanes 1–4) or anti-DJ-1 (lanes 5–8). RNA was radiolabeled, showing a smear of label in the DJ-1 IP samples (lane 5, bar on the left of the autoradiogram). A low concentration of RNase (lanes 2, 3, 6, and 7) did not remove label, but a high concentration (lane 8) resolved the DJ-1 samples to a single band of ≈35 kDa (lane 8, arrow). Markers on the right of the autoradiogram are in kDa. (e) M17 cells were transfected with WT (lane 2) or mutant (lanes 3–5) DJ-1 constructs containing a C-terminal V5-His tag. A vector containing GFP was used as a control (lane 1). DJ-1 was purified by using NiNTA beads and subjected to Western blotting with an anti-V5 antibody. (f) We performed qRT-PCR for selenoprotein genes, expressing these results as enrichment relative to GFP. As M26I is partially unstable, the amount of DJ-1 protein in the input and IP is lower, and thus all values were also corrected to amount of DJ-1 in the IP. Values are mean +/− SEM, n = 3–4 replicates per construct. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using one-way ANOVA for each gene with Bonferroni post hoc tests to compare mutants with WT DJ-1.

DJ-1 interacts directly with GG/CC-rich mRNA sequences. (a) Sequences recovered from CLIP tags for targets also present on the array; each of these was sequenced in duplicate. Similar sequences were seen in other sequences from the array dataset including GPx4 (5′ UTR) and MAPK8IP1 (both 5′ and 3′ UTR). (b) Consensus for all recovered CLIP tags. (c) In vitro binding of recombinant GST-tagged DJ-1 (7.5 nM) was assessed by pull down with biotinylated, single-stranded RNA (50 nM). DJ-1 was coprecipitated with the 5′-UTR of GPx4 (lane 2) but not the 3′-UTR (lane 3) or the coding sequence from UbC9 (lane 1). Data are representative of four independent experiments. (d) The 5′ UTR (lane 1) of GPx4 gives equivalent amounts of pull down compared with the full-length RNA (lane 2) and higher than the coding sequence of GAPDH (lane 3). (e) Diagrams of 5′ and 3′ reporter constructs, which were driven by a CMV promoter (P_CMV) and contained an SV40 poly adenylation [poly(A)] sequence. EGFP alone was used as a control for transfection. (f–j) M17 cell lines stably expressing nonspecific shRNA controls (lanes 1 and 2) or shRNA to DJ-1 (lanes 3 and 4) were transfected with reporter constructs where the 5′ UTR (f) or 3′ UTR (g) of GPx4 were cloned adjacent to the 5′ and 3′ end of EGFP. EGFP alone without any additional sequences is shown in h. Similar analyses were performed for the 5′ (i) and 3′ (j) UTR sequences from MAPK8IP1. Cell lysates were blotted for GFP and reprobed for β-actin, and quantitation is shown in the bar grpah below each pair of blots. All reporter construct assays were performed in two shRNA cell lines and each graph shows the average of at least three independent transfections per construct. *, P < 0.05; **, P < 0.01 by t test comparing lines with control shRNA and lines with DJ-1 shRNA.