Nurr1 3’UTR long variant is differentially regulated by miRNAs.

<p><b>A</b>. Schematic representation of reporter vectors used in the reporter gene assay. pEZX-MT01 is the control vector that codifies the reporter gene luciferase, pEZX-3’UTR short codifies luciferase fused to the Nurr1 3’UTR short and pEZX-3’UTR long codifies luciferase fused to Nurr1 3’UTR long. <b>B</b>. Dose/response curve of luciferase activity for pEZX-MT01 (control), pEZX-Nurr1-3’UTR short and pEZX-Nurr1-3’UTR long. <b>C</b>. Effect of miR-145, miR-302d or miR-130a overexpression on Nurr1 3’UTR short. <b>D</b>. Effect of miR-204, miR-93, miR-17, miR-302d, miR-455, miR-212 or miR-30a overexpression on Nurr1 3’ UTR long. <b>E</b>. Effect of miR-145or miR-130a overexpression on Nurr1 3’UTR long. <b>F</b>. Effect of miR-204 or miR-93 overexpression on Nurr1 3’UTR short. <b>G</b>. pR-let7 is a luciferase reporter vector with two sites for let-7a miRNA localized at the 3'UTR. The graph shows the effect of let-7a miRNA overexpression on pRL-let7a positive control vector. <b>B, C, D, E, F</b>. Forty eight hours after transfection the cells were harvested and luciferase activity and amount of proteins were measured. <b>C, D, E, F</b>. The results correspond to the average ± SEM of at least 3 independent experiments, performed each in triplicate. The nonparametric Mann-Whitney test was applied to determine statistical significance of the differences. *p<0.05 (Control versus specific miRNA); ***p<0.0001 (Control versus specific miRNA).</p

Nurr1 has multiple mRNA variants with different 3’UTR length.

<p><b>A.</b> Image from UCSC genome browser depicting the localization of Nurr1 (NR4A2) gene (first panel) in the rat genome (chromosome 3:38865326–38871768). Thick boxes correspond to exons, lines to introns and thin boxes to the 5’UTR on the left end, and to the 3’UTR on right end. The second panel shows PolyA-seq results obtained from brain and testis. The position of the lines indicates the end of the mRNA, and the height of the line indicates the relative abundance. The third panel shows the conservation of the NR4A2 gene sequence of rat compared to human and mouse genomes. <b>B</b>. Schematic representation of Nurr1 mRNA variants found in polyA-seq data. Aside from the rat Nurr1 variant already described in the databases, we identified 4 additional mRNA variants. The variant described in the databases was called Nurr1 3’UTR short (UTR length 658 bp), the variant highly expressed in brain was called Nurr1 3’UTR long (3’UTR length 1339 bp). The novel variant found in brain with lower expression was called Nurr1 variant 2 (3’UTR length 1084 bp). The variant highly expressed in testis was named Nurr1 variant 3 (3’UTR length 195) and the variant with the lower expression in testis that lacks 3’UTR and has a shorter seventh exon was named Nurr1 variant 4. <b>C</b>. The position of primer pairs to amplify the longest Nurr1 mRNA variant. <b>D.</b> RT-PCR products showing the expression of Nurr1 3’UTR long variant. The tissues studied were Hip: hippocampus; VTA: ventral tegmental area; SN: substantia nigra; H: heart; Sp: spleen and L: lung.</p

Bioinformatics search for miRNAs candidates to regulate the short or long Nurr1 variant.

<p><b>A.</b> Representation of the bioinformatics search for candidate miRNAs targeting Nurr1 short variant as a Venn diagram. The circles represent the parameters of the search: expression profiles and databases search. Gray numbers represent the interactions that were considered for the selection of miRNA candidates. <b>B</b>. Representation of the bioinformatics search for candidate miRNAs targeting Nurr1 long variant as a Venn diagram. For this search we only used only database search for candidate miRNAs. The circles represent tree different databases for miRNAs search: Targetscan, miRDB and microRNA.org. The coincidence of the three circles was considered for the selection of miRNAs. <b>C</b>. Schematic representation of Nurr1 3’UTR and miRNA seed region localization. The length of Nurr1 3’UTR long is 1339 bp and includes the Nurr1 3‘UTR short (658 bp), shown as white in the image, meanwhile the rest of the 3’UTR is shown in gray. The seed sites of miRNAs selected for the short 3’UTR are: miR-145 in nucleotide 25, miR-302d in nucleotide 465, and miR-130a in nucleotide 606. The seed sites of miRNAs selected for the specific part of the long 3’UTR are: miR-204 in nucleotide 870, miR-212 in nucleotide 1014, miR-93 and miR-302d in nucleotide 1063, miR-17 in nucleotide 1070, miR-455 in nucleotide 1177 and miR-30a in nucleotide 1279.</p

Reported miRNAs regulating Nurr1.

<p>Reported miRNAs regulating Nurr1.</p

Lysine 91 is the main SUMOylation site of Nurr1.

<p>(A) Schematic representation of full-length HA-Nurr1, and truncated isoforms HA-Nurr1ΔAF-2 (amino acids 1–353) and HA-Nurr1ΔAF-1 (amino acids 262–598). (B, C) Total extracts of COS-7 cells transfected with HA-Nurr1ΔAF-2 or HA-Nurr1ΔAF-1, plus Ubc9 and SUMO-2 were fractionated in SDS-PAGE and proteins analyzed with anti-HA (B) and anti-Nurr1 (C) antibodies. (D) Total extracts of COS-7 cells transfected with wild type HA-Nurr1 or the point mutants HA-Nurr1-K74R or HA-Nurr1-K91R were fractionated in SDS-PAGE. Western blot was performed with an anti-HA antibody.</p

Point mutant PIASγC342A fails to SUMOylate Nurr1.

<p>(A) Total extracts of COS-7 cells transfected with HA-Nurr1, SUMO-2 and PIASγ or the point mutant PIASγC342A were fractionated in SDS-PAGE and western blot assays performed with anti-HA (upper), anti-PIASγ (middle) and anti-SUMO-2 (bottom) antibodies. (B) HEK293cells were transfected with 100 ng of NBRE-3X-tk-LUC reporter and equimolar amounts of HA-Nurr1, PIASγ or PIASγC342A. Cells were harvested 48 hours post transfection and lysates assayed for luciferase activity. Results are expressed as fold of induction related to control (pcDNA3.1+) and correspond to the mean ± S.E.M. of three independent assays performed each in triplicate. Statistical significance was estimated by the non-parametric Mann-Whitney U test. *p<0.05 (Nurr1+PIASγ v/s Nurr1) and (Nurr1+ PIASγC342A v/s Nurr1). In the bottom, western blots showing the expression of recombinant proteins and actin used as loading control.</p

Lysine 91 of Nurr1 is in a synergy control (SC) motif.

<p>(A) Consensus SC motif <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055035#pone.0055035-IiguezLluh1" target="_blank">[14]</a> and putative Nurr1 SC motif. (B) HEK293 cells were transfected with 100 ng of 1NBRE-Luc (one NBRE element), 3NBRE-Luc (three NBRE elements) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055035#pone.0055035-Woronicz1" target="_blank">[15]</a> or NBRE-3X-tk-LUC (three NBRE elements) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055035#pone.0055035-Castro1" target="_blank">[5]</a> reporters and equimolar amounts of HA-Nurr1 or point mutant HA-Nurr1-K91R. Cells were harvested 48 hours post-transfection and lysates assayed for luciferase activity. Results are expressed as fold of induction related to control (pCGN, empty vector) and correspond to the mean ± S.E.M. of three independent assays performed each in triplicate. Scale in left axis is for 1NBRE-Luc and 3NBRE-Luc reporters' activity, and scale in right axis is for NBRE-3X-tk-LUC reporter activity. Statistical significance was estimated by the non-parametric Mann-Whitney U test *p<0.05 (HA-Nurr1-K91R v/s HA-Nurr1). (C) HEK293cells were transfected with 100 ng of 1NBRE-Luc or 3NBRE-Luc reporters and equimolar amounts of HA-Nurr1ΔAF-2 or point mutant HA-Nurr1ΔAF-2-K91R. Cells were harvested 48 hours post-transfection and lysate assayed for luciferase activity. Results are expressed as fold of induction related to control (pCGN) and correspond to the mean ± S.E.M. of three independent assays performed each in triplicate.</p

SUMOylation on lysine 91 does not modify Nurr1 half-life and location.

<p>(A) HEK293 cells were transfected with HA-Nurr1 or HA-Nurr1-K91R. Twelve hours after transfection the cells were treated with cycloheximide and harvested at the indicated hours. Total extracts were fractionated in SDS-PAGE and western blots developed with anti-HA and anti-actin (load control) antibodies. Densitometric analysis of 3 independent experiments was performed with Image J software. Data is expressed as percentage of HA-Nurr1 or HA-Nurr1-K91R expression at cero time and correspond to the mean ± S.E.M. (B) Representative western blots of HA-Nurr1 and HA-Nurr1-K91R during cycloheximide treatment. (C) PC12 cells were transfected with the indicated plasmids. Fixed cells were subjected to double immunofluorescence protocols using HA and PIASγ antibodies. Alexa 594 (red) second antibody was used to visualize HA and Alexa 488 (green) second antibody was used to visualize PIASγ. Cells were examined by deconvolution microscopy. (D) Colocalization of Nurr1 or the mutant Nurr1-K91R with PIASγ using van-Steensel analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055035#pone.0055035-vanSteensel1" target="_blank">[19]</a>.</p

PIASγ requires the ₂₀LXXLL₂₄ motif to interact, SUMOylate and repress Nurr1.

<p>(A) Schematic representation of full-length PIASγ indicating functional domains SAP: <i>Scaffold Attachment factor-A/B acinus and PIAS</i>; the PINIT domain; SP<i>-</i>(<i>Siz/PIAS</i>)<i>-</i>RING and SIM: <i>SUMO Interaction Motif</i>. PIASγ has two LXXLL motifs. Mutations changing leucines (L) by alanines (A) in each LXXLL domain are depicted for GST-PIASγmut1 and GST-PIASγmut2. (B) GST, GST-PIASγ, GST-PIASγmut1 and GST-PIASγmut2 retained in glutathione-agarose beads were incubated with extracts from COS-1 cells transfected with Myc-Nurr1_363–598. Retained proteins were fractionated in SDS-PAGE and western blot developed with anti-Myc monoclonal antibody, revealing that only GST-PIASγ and GST-PIASγmut2 are able to interact with Nurr1. Recombinant GST-PIASγ proteins were equally loaded in each GST-pull down assays as shown by western blot using anti-PIASγ antibody (bottom). (C) Luciferase reporter assay showing that full-length PIASγmut1 lose repressor capacity over Nurr1 transactivity. HEK293 cells were transfected with the NBRE-3X-tk-LUC along with HA-Nurr1, PIASγ or PIASγmut1. After 48 hours, cells extracts were assayed for luciferase activity. Data are expressed as percentage of Nurr1 transactivation and correspond to the mean ± S.E.M of 4 independent experiments each performed in triplicates. Statistical significance was estimated by the non-parametric Mann-Whitney U test. *p<0.05 (Nurr1+PIASγ v/s Nurr1+PIASγmut1). In the bottom, western blots showing the expression of recombinant proteins and actin used as loading control. (D) Total extracts of COS-7 cells transfected with HA-Nurr1, SUMO-2 and PIASγ or PIASγmut1 were fractionated in SDS-PAGE and western blots performed with anti-HA, anti-PIASγ and actin (load control) antibodies.</p

Nurr1 is SUMOylated by SUMO-2.

<p>(A) Schematic representation of Nurr1 (bottom) showing the position of four putative SUMOylation sites according with SUMOplot^TM software analysis (middle). The table shows the sequences of the potential SUMOylation sites of rat Nurr1 sorted from the highest score. Putative SUMO acceptor lysines (K) are highlighted, and potential SUMO sites are underlined. (B) COS-7 cells were transfected with plasmids expressing HA-Nurr1, Ubc9, and SUMO-1, SUMO-2 or SUMO-3. Cells were harvested 48 hours post-transfection and lysed directly in loading buffer containing the SUMO-isopeptidase inhibitor N-ethylmaleimide 20 mM, and fractionated in SDS-PAGE. Representative western-blot with anti-HA (upper) and anti-SUMO-2 (bottom) antibodies. (C) Quantitative densitometry analysis of Nurr1-SUMO-2 signal described in (B), using Image J software. Data correspond to the mean ± S.E.M. of 3 independent experiments for each condition. Statistical significance was estimated by the non-parametric Mann-Whitney U-test. * p<0.05 (Nurr1+Ubc9+SUMO-2 v/s Nurr1). (D) Total lysates from COS-7 cells transfected with HA-Nurr1, Ubc9 and SUMO-2 were immunoprecipitated with an anti-Nurr1 antibody or control IgG. The immunoprecipitates were analyzed in western blots with anti-HA antibody. Bands for immunoglobulin are indicated as IgG heavy chain (H).</p