1. Volume 55, Issue 2, Pages 186-198 (July 2014)
Direct Binding of Ataxin-2 to Distinct Elements in 3′ UTRs Promotes mRNA Stability and Protein Expression 
Moe Yokoshi, Quan Li, Munetaka Yamamoto, Hitomi Okada, Yutaka Suzuki, Yukio Kawahara 
Molecular Cell 
Volume 55, Issue 2, Pages (July 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 55, 186-198DOI: (10.1016/j.molcel.2014.05.022)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 RNA Targets of Human Ataxin-2 Identified by PAR-CLIP
(A) Schematic diagram of human Ataxin-2 tagged with Halo at the N terminus. PolyQ, polyglutamine tract; LSm, LSm domain; LSmAD, LSm-associated domain; PAM2, PABPC1-interacting motif-2.
(B) Phosphorimages of SDS gels that resolved immunoprecipitated Ataxin-2 wild-type and the ΔPAM2 deletion mutant, both of which were crosslinked with radiolabeled RNA fragments. The concentration of MNase used for RNA fragmentation is indicated at the top. See also Figure S1.
(C) The number of mismatches (left panel) and the frequency of each nucleotide mismatch (right panel) in PAR-CLIP reads aligned to human genomic sequence (hg19) are shown for Ataxin-2 wild-type and the ΔPAM2 deletion mutant. See also Table S1.
(D) Distribution of the binding sites of Ataxin-2 wild-type (upper panel) and the ΔPAM2 deletion mutant (lower panel) in different types of RNA (left panels) and in pre-mRNAs (right panels). See also Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Analysis of Ataxin-2-Binding Sites
(A) A Venn diagram that shows the number of overlapping and nonoverlapping genes targeted by Ataxin-2 wild-type (WT) and the ΔPAM2 deletion mutant.
(B) A representative RNA recognition motif of Ataxin-2 inferred by cERMIT. See also Table S4.
(C) Analysis of the number of AUUUN sequences around the crosslinked sites for Ataxin-2 WT.
(D and E) Results of the in vitro binding assay with the indicated amounts of Ataxin-2 WT or the ΔPAM2 deletion mutant and radiolabeled RNA that contained the Ataxin-2-target sites found within the 3′ UTR of ARL6IP1 (D) or FAM199X (E). See also Figure S2.
(F) Results of the in vitro binding assay with the indicated amounts of Ataxin-2 WT or the ΔPAM2 deletion mutant and radiolabeled short RNA (24 nt) that contained a single AUUUA or ACCCA sequence.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Analysis of mRNA Stability and Protein Abundance after the Perturbation of Ataxin-2 Expression
(A and B) The expression of individual mRNAs was quantified systematically by microarray 48 hr after depletion (A) or overexpression (B) of Ataxin-2. Then, the cumulative distributions of changes in the stability of the mRNAs in the indicated categories were calculated. The change in expression of each mRNA is defined as the mean value of three independent experiments. The category “Target” includes all the genes that harbor binding sites for the Ataxin-2 ΔPAM2 deletion mutant in the 3′ UTR that had been identified by PAR-CLIP analysis. The categories “Top 20%” and “Top 50%” contain the highest-ranked genes on the basis of the number of binding sites identified by PAR-CLIP analysis. The category “Non-target” includes all the genes that are not targeted by Ataxin-2. The category “Strict non-target” includes the genes that harbor no possible binding sites for Ataxin-2 in PAR-CLIP analysis, regardless of the number of reads and T-to-C conversions. The number of genes in each category and the p values calculated between each category and the category “Strict non-target” are indicated in parentheses.
(C) Values represent the half-life of each mRNA as quantified by the addition of actinomycin D after transfection of either Ataxin-2 siRNA or control siRNA (mean ± SEM; n = 3; ∗p < 0.05). See also Figure S3.
(D) The change in protein expression of Ataxin-2 targets at 48 hr after depletion of Ataxin-2 was detected by immunoblot analysis with the indicated antibodies. Values represent the relative expression of the indicated proteins normalized to GAPDH (mean ± SEM; n = 3; ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Comparative Analysis of RNA Targets between Ataxin-2 and HuR
(A and B) A Venn diagram that shows the number of overlapping and nonoverlapping genes (A) and clusters (B) targeted by Ataxin-2 and HuR. See also Figure S4.
(C) A Venn diagram that shows the number of overlapping and nonoverlapping AUUUN recognition motifs targeted by Ataxin-2 and HuR. A motif is defined as an AUUUN sequence that contains a crosslinking site with at least one T-to-C conversion within the AUUUN sequence plus an additional 5 nt upstream and downstream of the motif.
(D) The relative location and number of clusters bound to Ataxin-2 (red) or HuR (blue) in the 3′ UTR of mRNAs are plotted.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
The Effect of Ataxin-2 Binding to the 3′ UTRs of mRNA on mRNA Stability and Protein Expression
(A) Schematic structures of the reporter constructs used for the λN tethering assay. Five tandem boxB hairpins (5× boxB highlighted in pink), which can be bound by λN-fusion proteins (yellow) with high affinity, were inserted into the 3′ UTR of a Renilla luciferase (Renilla-Luc highlighted in blue) reporter construct. A firefly luciferase (Firefly-Luc highlighted in green) reporter construct was cotransfected with the Renilla-Luc reporter construct as a reference.
(B) The results of the tethering assay with a Renilla-Luc reporter with or without boxB hairpins and Halo-λN, Halo-λN-TNRC6A, or Halo-λN-Ataxin-2 wild-type (WT) protein are shown. Values represent relative Renilla-Luc activities normalized to Firefly-Luc activities (n = 4; ∗p < 0.05). The relative Renilla-Luc activity obtained with the reporter with boxB hairpins and Halo-λN protein is set as one. Error bars represent the SEM. See also Figure S5.
(C and D) After Renilla-Luc reporter mRNA with (C) or without (D) boxB hairpins had been induced for 2 hr with Dox, the effect of tethering either Halo-λN-Ataxin-2 WT or Halo-λN on expression of the reporter protein was monitored by measuring Renilla-Luc activity at the indicated time points. Values represent relative Renilla-Luc activities normalized to the number of cells with the relative Renilla-Luc activity obtained with Halo-λN protein at 8 hr after induction set as one (mean ± SEM; n = 3; ∗p < 0.05).
(E) After Renilla-Luc reporter mRNA with or without boxB hairpins had been induced for 2 hr with Dox, the effect of tethering either Halo-λN-Ataxin-2 WT or Halo-λN on the stability of the reporter mRNA was monitored at the indicated time points. Values represent the relative amount of Renilla-Luc mRNA normalized to GAPDH mRNA (mean ± SEM; n = 3; ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
The Effect of Partial Deletion and PolyQ Expansion on mRNA Stability and Target Protein Expression
(A) The expression of Halo-λN-Ataxin-2 WT and the deletion mutants indicated at the top was confirmed by immunoblot analysis with an anti-Halo antibody. The expression of GAPDH is shown as a reference. LSmAD1 encompasses the N-terminal half of LSmAD (aa 353–409), and LSmAD2 encompasses the C-terminal half of LSmAD (aa 429–477). See also Figure 1A.
(B and C) Results of the tethering assay with a Renilla-Luc reporter with boxB hairpins and the Halo-λN-tagged Ataxin-2 deletion mutants indicated at the bottom. Significant reductions in the relative Renilla-Luc activities obtained with the deletion mutants compared to that obtained with the WT are indicated by a hash (mean ± SEM; n = 4; #p < 0.05). See also Figure S6.
(D) After Renilla-Luc reporter mRNA with boxB hairpins had been induced for 2 hr with Dox, the effect of tethering either Halo-λN-Ataxin-2 WT or the deletion mutants indicated at the bottom on the stability of the reporter mRNA was monitored at 24 hr postinduction. Values represent the relative amount of Renilla-Luc mRNA normalized to GAPDH mRNA with the relative amount of Renilla-Luc mRNA obtained with Halo-λN protein set as one (mean ± SEM; n = 3; #p < 0.05).
(E) After Renilla-Luc reporter mRNA with boxB hairpins had been induced for 2 hr with Dox, the effect of tethering either Halo-λN-Ataxin-2 WT or the deletion mutants indicated at the bottom on expression of the reporter protein was monitored by measuring Renilla-Luc activity at 24 hr postinduction. Values represent relative Renilla-Luc activities normalized to the number of cells with the relative Renilla-Luc activity obtained with Halo-λN protein set as one (mean ± SEM; n = 3; #p < 0.05).
(F) The expression of Halo-λN-Ataxin-2 WT and the mutants with an expanded polyQ tract was detected by immunoblot analysis with an anti-Halo antibody. The expression of GAPDH is shown as a reference.
(G) Results of the tethering assay with a Renilla-Luc reporter with boxB hairpins and Halo-λN-Ataxin-2 WT or the mutants with an expanded polyQ tract are shown. Values represent relative Renilla-Luc activities normalized to Firefly-Luc activities. The relative Renilla-Luc activity obtained with the reporter with boxB hairpins and Halo-λN protein is set as one. Significant reductions in the relative Renilla-Luc activities obtained with the mutants compared to that obtained with the WT is indicated by a hash (mean ± SEM; n = 4; #p < 0.05). See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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