Supplemental Methods

Microarray analysis

Immunoprecipitated mRNA was collected as described above and hybridized to the Atlas Human apoptotic cDNA microarray membrane (Clontech). Gene hybridization levels were compared between samples to determine those messages which increase following 3A2 IP, relative to IP with IgG control antibody.

Northern blotting

Northern blot probes were synthesized by PCR reaction off of template cDNA. HuR, GAPDH and 18S probes have been previously described 1. GFP template (Clontech) was amplified using the following primers: GFP-F: 5’-AGTGCTTCAGCCGCTACCC-3’ and GFP-R: 5’-GCTTGTGCCCCAGGATGTT-3’. Caspase-9 template (Open Biosystems) was amplified using the following primers: C9-Forward: 5’-GGGAGCAGAAAGACCATGGGT -3’ and C9-Reverse: 5’-ATTTGCGGCCGCTTATGATGTTTTAAAGAAAAGTT-3’. ProT template (generous gift from T. Kobayashi 2) was amplified using the following primers: ProTalpha-F: 5’-GTAGACACCAGCTCCGAAATCAC-3’ and ProTalpha-F: 5’-TCTGCTTCTTGGTATCGACATCG-3’.

RT-PCR and RT-qPCR

Primers used for PCR to detect caspase-9, hnRNPA 1 and ProT DNA from IP/RT experiments were synthesized by Integrated DNA Technologies (IDT), and were the following: caspase-9-Forward: 5’-GCATTTCATGGTGGAGGTG-3’, caspase-9-Reverse: 5’-GTAGGACACAAAGATGTCAC-3’; ProT-Forward: 5’-CCAACCCAAACCATGAGAA-3’, ProT-Reverse: 5’-GGTCACACCACAAGTAAAGTCAG-3’; hnRNPA1-Forward: 5’-AAGGGGCTTTGCCTTTGTAACCTT-3’, hnRNPA1-Reverse: 5’-CATTATAGCCATCCCCACTGCC-3’. Primers used for qPCR to detect caspase-9 and ProT were also synthesized by IDT and were: qcasp9-Forward: 5’-GTTTGAGGACCTTCGACCAG-3’, qcasp9-Reverse: 5’-GCATTAGCGACCCTAAGCAG-3’; qProT-Forward: 5’-CTGCTAACGGGAATGCTGA-3’, qProT-Reverse: 5’-TCGACATCGTCATCCTCATC-3’

FLAG-caspase-9 plasmid production

The synthesis of FLAG-caspase-9 full length and FLAG-caspase-9 mut AREsplasmids was performed as follows. Caspase-9 sequence was amplified by PCR, using full-length caspase-9 cDNA as the template. A BamHI site was created at the 5’-end of the PCR product, followed by nucleotides encoding for a FLAG tag, and a NotI restriction site was created at the 3’-end. The primers used for full-length caspase-9 were: FLAG-C9-F: 5’-GCAGGATCCATGGACTACAAAGACGACGACGACAAAGACGAAGCGGATCGGCGGCTC -3’ and full-C9-R: 5’- ATTTGCGGCCGCTTTTAAAATTAATGCAATATA-3’. The PCR fragment was cloned into the BamHI/NotI sites of pcDNA3 vector (GE Healthcare). To generate the mutated AREs caspase-9, the same forward primer was used, with a reverse primer which produced multiple mutations in ARE2 (shown in Figure 2B). This primer was: mut-C9-R: 5’- ATTTGCGGCCGCTTTTAAAATTAATGCGGTGTGGGGGAAGTTGGCGGTTTTTGGGGTTTCACTTAGAATTCA-3’. The mutations of the indicated nucleotides in ARE1 in this plasmid were then performed by NorClone Biotech Laboratories (London, Ontario, Canada).

Gel-shift

Two synthetic oligonucleotide caspase-9 ARE cRNA probes were generated [spanning nucleotides 1841-1870 (ARE-1) and 1944-1988 (ARE-2) of the 3’ UTR of caspase-9]. Each was fused to a T7 promoter and transcribed in vitro as previously described 3. 50 000cpm of purified [32P]UTP-labelled cRNA was then incubated with 500ng of either recombinant GST, GST-HuR4, GST-HuR-CP1 or GST-HuR-CP2 protein5 and RNA-binding assay was performed as done previously 6. Two additional probes were generated in which every uracil ribonucleotide of these probes was mutated to a cytosine (mut-ARE-1 and mut-ARE-2), other than those in the T7 promoter region.

Kd determination by Gel-shift

Gel shift was performed as described above using 10 000cpm of purified [32P]UTP-labelled ARE2 cRNA probe and varying concentrations of GST-HuR, GST-HuR-CP1 and GST-HuR-CP2. Band intensities were quantified, and plots of bound/unbound signal against concentration of GST-protein were generated. The slope of linear trendlines from these graphs were used to determine experimental Kd values.

Polysome fractionation and Slot blot

HeLa cells were treated as described previously 7. Briefly, cells were collected and homogenized, and then centrifuged and fractionated using a Teledyne ISCO density gradient fractionation system (Foxy JR fraction collector with a UA-6 UV detector). Each fraction was collected from 22 drops, and 24-28 fractions were collected for each sample. To isolate proteins from each fraction, overnight precipitation was done at -20°C with two volumes of ethanol. RNA was extracted following the same protocol used to isolate mRNA from the IPs described above, and samples were then analyzed by slot blot 8

References for Supplemental Mathods

1.Di Marco S, Mazroui R, Dallaire P, Chittur S, Tenenbaum SA, Radzioch D et al.NF-(kappa)B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release. Mol Cell Biol 2005; 25(15): 6533-45.

2.Kobayashi T, Wang T, Maezawa M, Kobayashi M, Ohnishi S, Hatanaka K et al. Overexpression of the oncoprotein prothymosin alpha triggers a p53 response that involves p53 acetylation. Cancer Res 2006; 66(6): 3137-44.

3.Gallouzi IE, Parker F, Chebli K, Maurier F, Labourier E, Barlat I et al. A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability. Mol Cell Biol 1998; 18(7): 3956-65.

4.Brennan CM, Gallouzi IE, Steitz JA. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol 2000; 151(1): 1-14.

5.Beauchamp P, Nassif C, Hillock S, van der Giessen K, von Roretz C, Jasmin BJ et al. The cleavage of HuR interferes with its transportin-2-mediated nuclear import and promotes muscle fiber formation. Cell Death Differ 2010.

6.Di Marco S, Hel Z, Lachance C, Furneaux H, Radzioch D. Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA. Nucleic Acids Res 2001; 29(4): 863-71.

7.Cammas A, Pileur F, Bonnal S, Lewis SM, Leveque N, Holcik M et al. Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol Biol Cell 2007; 18(12): 5048-59.

8.Lian XJ, Gallouzi IE. Oxidative Stress Increases the Number of Stress Granules in Senescent Cells and Triggers a Rapid Decrease in p21waf1/cip1 Translation. J Biol Chem 2009; 284(13): 8877-87.

Supplemental Figure Legends

Supplemental Figure 1: HuR associates with multiple apoptotic-related mRNAs

HeLa cells were lysed and subjected to immunoprecipitation using anti-HuR antibody, or IgG control. The mRNAs isolated were hybridized to the Atlas Human apoptotic array. Analysis of the arrays provided a list of messages which showed to associate with HuR two-fold or more by immunoprecipitation, compared to IgG control antibody. mRNAs that associated with the HuR antibody by two-fold or more (as defined by the signal ratio on the arrays between anti-HuR and -IgG control IPs) were tabulated.

Supplemental Figure 2: The 3’UTR of caspase-9 mRNA contains two AU-rich elements

The 3’ UTR of caspase-9 mRNA was found as displayed as NCBI Reference Sequence NM_001229.2. The two AU-rich elements contained in this sequence are underlined and in bold font, representing the regions of nucleotides 1841 to 1870 and 1944 to 1988.

Supplemental Figure 3:Knockdown of HuR decreases caspase-9 protein expression in MEFs

(A)MEFs cells were treated with siRNA against HuR or control siRNA for 48h, and their total protein was collected and analyzed by western blot, using antibodies against caspase-9, HuR and α-tubulin (loading control). (B)Quantification of band intensities was performed using ImageQuant software. The asterisk indicates a significant difference in mean values. The blot shown is representative of three independent experiments.

Supplemental Figure 4: The AREs of the casp-9 3’UTR regulate mRNA stability

This graph is a different presentation of the data shown in Figure 3F. Here, the values are graphed on a logarithmic (base 10) scale, instead of a linear scale.

Supplemental Figure 5: MG132 inhibits proteosome activity

293T cells stably expressing a proteosome targeting signal, tagged with GFP (GFPμ) were treated with MG132 in parallel to samples described in Figure 4C. Lysates from these cells were analyzed by western blot probing for GFP and G3BP (loading control), and a representative image from three independent experiments is shown.

Supplemental Figure 6: The effect of overexpressing HuR and its CPs on caspase-9 and ProT mRNA

The effect of overexpressing GFP-HuR mutants. (A) Total mRNA was extracted from HeLa cells 48 hours after having been transfected with GFP-plasmid alone, or a plasmid containing GFP-HuR, -HuR-CP1 or HuR-CP2. The mRNA was analyzed by northern blot, probing for ProT, caspase-9 and GAPDH (loading control).(B)Shown is a representative northern of three independent experiments. The levels of mRNA in each lane were quantified, and the levels are shown as the mean with errors bars representing the SEM. Statistical analysis indicated that all differences in RNA levels are not significant.

Supplemental Figure 7: HuR-CPs and full-length HuR can rescue Bax-induced cell death

(A)HeLa cells were transfected with siRNA against HuR (or control siRNA) as well as with HA-Bax plasmid. 32h later, cells were provided with 50nM AP-GST, AP-HuR-GST, or 25nM AP-HuR-CP1-GST and 25nM AP-HuR-CP2-GST. Cells were photographed 16 hours later, and a representative phase image of these cells are shown, which is representative of three different fields of view, each from two independent experiments.(B)The relative percentage of remaining cells was determined by counting the number of cells present following the treatments described above from all fields of view, and expressing this as a percentage compared to the number of cells present on the samples treated with siHuR and AP-GST. Error bars represent the SEM of two independent experiments.

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