hnrnp C promotes APP translation by competing with FMRP for APP mrna recruitment to P bodies

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1 hnrnp C promotes APP translation by competing with for APP mrna recruitment to P bodies Eun Kyung Lee 1, Hyeon Ho Kim 1, Yuki Kuwano 1, Kotb Abdelmohsen 1, Subramanya Srikantan 1, Sarah S. Subaran 2, Marc Gleichmann 3, Mohammed R. Mughal 3, Jennifer L. Martindale 1, Xiaoling Yang 1, Paul F. Worley 4, Mark P. Mattson 3 & Myriam Gorospe 1,* 1 Laboratory of Cellular and Molecular Biology, NIA-IRP, NIH, Baltimore, MD 21224, USA 2 Confocal Imaging Facility, Research Resources Branch, NIA-IRP, NIH, Baltimore, MD 21224, USA 3 Laboratory of Neurosciences, NIA-IRP, NIH Baltimore, MD, 21224, USA 4 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 225, USA

2 Lee et al., Supplementary Fig. 1 a b (sirna) APP β-actin 6 IgG HuR HuD AUF1 TIA-1 TIAR RCK NCL (fold) (SD) c APP mrna in IP (Fold) (sirna) (IP) p=.47 hn RNPC hnrnp p=.4 APP mrna (Fold enrichment) d (sirna) (Input) (IP) hn RNPC (IP) IgG HuR HuD AUF1 TIA-1 TIAR RCK NCL (WB) hrnpc Supplementary Figure 1. RBPs are involved in the regulation of APP expression. (a) After individually silencing the indicated RBPs in BE2-M17 human neuroblastoma cells, the levels of APP and loading control β-actin were measured by Western blot analysis. (b) RNP immunoprecipitation (IP) analysis to study the enrichment of APP mrna in samples obtained after IP of the RBPs shown. Analysis was performed as explained in Figure 1a (main paper). In (a) and (b), the data are the means +s.d. of three independent experiments. (c) BE2-M17 cells were transfected with the sirnas indicated and cell lysates were collected 48 h later; RNP IP analysis using antibodies that recognized or was followed by RT-qPCR detection of APP mrna. Data are the means +s.d. from three experiments. (d) The abundance of hnrnp C and in Input (1 µg) and in the IP samples (Methods) was detected by Western blot analysis.

3 a APP mrna Lee et al., Supplementary Fig. 2 CR-C G-quartet domain bp (1) 72 bp (2) 112 bp (3) 48 bp (4) 69 bp (5) 89 bp (6) b GAPDH UTR IN c APP mrna NM_ UTR Coding region (CR) 3 UTR A B C D E F G H IN A B C D E F G H GAPDH 3 UTR HuR HuD Supplementary Figure 2. RBPs interacting with the APP 3 UTR. (a) schematic of the APP mrna depicting segment C of the coding region and additional segments (1-6) that were tested by biotin pulldown assays and the nucleotide positions spanned by each fragment. (b) After incubation with the biotinylated RNAs indicated, the association of RBPs hnrnp C and was tested by Western blot analysis; biotinylated GAPDH 3 UTR was included as negative control. IN, input (1 g lysate). RBPs HuR and HuD associate with the APP mrna. (c) Top, schematic of the APP mrna depicting the 5 UTR, CR, and 3 UTR, as well as the different segments that were tested by biotin pulldown assays and the nucleotide positions that each fragment spans. Bottom, after incubation with the biotinylated RNAs indicated, the association of RBPs HuR and HuD was tested by Western blot analysis; Biotinylated GAPDH 3 UTR was included as negative control. Antibodies recognizing HuR and HuD were from Santa Cruz Biotech.

4 Lee et al., Supplementary Fig. 3 WCE CE NE H A H A H A α-tubulin β-actin lamin B Supplementary Fig. 3. Subcellular localization of RBPs. BE2-M17 cells were incubated with 1 µm of hydrogen peroxide (H) or with 5 µm of sodium arsenite (A) for 1 h. Cells were then lysed in the presence of 2 µg ml -1 digitonin to fractionate cytosolic (CE) and nuclear (NE) components; 1 µg protein lysate were assayed per lane.

5 Lee et al., Supplementary Fig. 4 4S 6S 8S Puromycin LMWP HMWP Direction of sedimentation Percent APP mrna 4 sirna sirna sirna sirna Percent β -actin mrna Gradient fraction hnrnp C sirna A sirna Gradient fraction Gradient fraction Supplementary Figure 4. APP mrna is associated with polysomes. Analysis of the distribution of APP and β-actin mrnas studied in the same three sirna transfection groups as in Figure 2d, except that the cells were incubated with Puromycin (2 µm for 1 h) before lysis. Compared with the data shown in Figure 2d (where distributions peaked at fraction 1), disruption of the polysomes by treatment with Puromycin triggered robust shifts (leftward) to lower molecular weight fractions for all mrnas, peaking at fractions 5-6. These data indicate that the APP and βactin mrnas in Figure 2d were associated with translating polysomes.

6 Lee et al., Supplementary Fig APP mrna (fold) (sirna) 25 ng 5 ng 1 µg 2 µg Supplementary Figure 5. Analysis of the linearity of RT-qPCR reactions. BE2- M17 cells were transfected with the sirnas indicated and total RNA was prepared 48 h later. Different amounts of RNA were used in reverse transcription reactions, followed by quantitative, real-time PCR amplification (qpcr). As shown, regardless of the amount of starting material, the relative levels of APP mrna among the three groups ( sirna, sirna, sirna) were comparable, indicating that RT-qPCR was a suitable method to analyze APP mrna levels in these cell populations.

7 Lee et al., Supplementary Fig. 6 RCK Ago1 Ago2 sirna GFP- APP GFP- -actin Supplementary Figure 6. does not repress APP translation in the absence of PB components. After silencing individually RCK, Ago1 or Ago2, GFP- was overexpressed in BE2-M17 cells. APP expression levels were assessed by Western blot analysis.

8 Lee et al., Supplementary Fig. 7 a c APP mrna in IP (Fold) * IgG RCK IgG RCK pgfp pgfp- b pms2-app + sirna Merge MS2-YFP/APP-MS2 RCK DIC Merge Supplementary Figure 7. Colocalization of tagged APP mrna with the PB protein RCK. (a) Twenty-four h after transfection of pegfp or pgfp-, the presence of APP mrna in RCK IP samples was measured by RNP IP using anti-rck or control IgG antibodies, followed by RT-qPCR analysis. Data represent the means +s.d. from three independent experiments. *, p<.5. (b,c) Plasmids pms2-app and pms2-yfp (described in Fig. 7a), were used to study the localization of the APP RNA (segment C) in BE2-M17 cells. Signals were detected by confocal microscopy. (b) By 48 h after transfecting sirna, MS2-APP RNA was visualized using MS2-YFP (green fluorescence), RCK signals were detected by immunofluorescence (red), and their colocalization was assessed (Merge, yellow). DIC, differential interference contrast. (c) Cells were incubated with anti-rck antibody and fluorescence signals were visualized with by confocal microscopy (Z-sectioning mode). Bottom, Z-sections show colocalization (yellow) of MS2-YFP/MS2-APP (green) with RCK (red).