1. Volume 51, Issue 6, Pages 737-750 (September 2013)
Human Inositol Polyphosphate Multikinase Regulates Transcript-Selective Nuclear mRNA Export to Preserve Genome Integrity 
Vihandha O. Wickramasinghe, Jane M. Savill, Sreenivas Chavali, Asta B. Jonsdottir, Eeson Rajendra, Tamara Grüner, Ronald A. Laskey, M. Madan Babu, Ashok R. Venkitaraman 
Molecular Cell 
Volume 51, Issue 6, Pages (September 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 51, 737-750DOI: (10.1016/j.molcel.2013.08.031)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
IPMK Depletion or Catalytic Inactivation Triggers the Nuclear Accumulation of Poly(A)+ RNA in Human Cells
(A) RNA FISH showing nuclear accumulation of poly(A)+ RNA in IPMK-, NXF1-, ALY-, and GANP-depleted Cal51 cells 72 hr posttransfection. Images are representative of five independent experiments. Scale bar, 5 μm. Western blots confirm protein depletion.
(B) A ratio of nuclear to cytoplasmic poly(A)+ RNA intensity was taken per cell for ≥1,000 cells/sample as described. Line graphs plotted for different samples represent cumulative values from five independent experiments. An arbitrary value of 1 is given to siControl.
(C) An averaged representation of nuclear to cytoplasmic ratios of poly(A)+ RNA as plotted in (B). Values represent the mean of duplicate readings from five independent experiments, ± SEM.
(D) Superposition (right) of the crystal structures of human IP3-K (left) and yeast Ipk2 (middle). Residues predicted to be essential for cofactor binding are indicated, with the corresponding residue numbering for human IPMK in brackets.
(E) The ratio of nuclear to cytoplasmic poly(A)+RNA intensity measured as described above in (B) is shown for cells expressing different catalytic mutants of IPMK fused to GFP. Values represent the mean of duplicate readings from five independent experiments, ± SEM. Statistically significant pairwise comparisons are indicated (one-way ANOVA, followed by Dunnett’s post test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
IPMK Depletion Decreases the Nuclear Export of a Subset of Human Transcripts
(A) Microarray analysis tabulates the number of genes downregulated in the cytoplasmic fraction from IPMK-depleted cells compared to controls. Functional annotation of genes downregulated in the cytoplasmic fraction from IPMK-depleted cells identifies transcripts enriched for proteins with the indicated Gene Ontology terms (p < 10−21). Derivation of p values is detailed in the Experimental Procedures.
(B) mRNAs encoding DNA repair proteins were quantitated by qRT-PCR in cytoplasmic RNA extracted from Cal51 cells treated with siIPMK(D2) or siControl. Plots are relative to GAPDH expression in siControl-treated cells, assigned an arbitrary value of 1, and show the mean of triplicate readings from three independent experiments, ± SEM.
(C) Protein expression analysis of genes affected (or unaffected) by IPMK(D2) depletion, without or with MMC (50 ng/ml) for 24 hr. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
IPMK Depletion Selectively Blocks the Nuclear Export of mRNAs Encoding HR, but Not NHEJ Proteins
(A) mRNA localization was visualized by hybridization of control siRNA-treated or IPMK-depleted cells with Cy3-labeled probes. Scale bar, 5 μm.
(B) Quantitation of images in (A) showing the average nuclear to cytoplasmic intensity of mRNA per cell in IPMK-depleted cells normalized to siControl. Plots represent the mean from three independent experiments, n > 100 cells per replicate, ± SEM. siControl is assigned a value of 1.
(C) Scanning analysis of transcript intensities was performed using ImageJ. Nuclei used for scanning and the scanning axis are indicated by thicker white lines in (A). Pairs of nuclei of same scan width as determined by DAPI staining were used. Nucleus and cytoplasm (Cyt) are indicated.
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6. Figure 4
IPMK Depletion Suppresses the Accumulation of RAD51, but Not γH2AX, at Sites of DNA Damage
(A) RAD51 and γH2AX foci formation in BJ-5ta cells treated with control or IPMK siRNAs, before exposure to MMC for 24 hr. Images show representative fields for DNA (blue), RAD51 (green), and γH2AX (red) staining. Scale bar, 5 μm.
(B) RAD51 (top) and γH2AX (bottom) foci formation in three different cell lines treated with control or IPMK siRNAs, before exposure to MMC for 24 hr. Results are normalized to siControl, assigned an arbitrary value of 1. Plots represent the mean of triplicate repeats from three representative experiments, ± SEM.
(C) Similar analyses in Cal51 cells exposed to 10Gy ionizing radiation, or for 24 hr to the indicated concentrations of MMC or carboplatin. Plots represent the mean of triplicate repeats from three representative experiments, ± SEM.
(D) Cells overexpressing different catalytic mutants of IPMK fused to GFP were exposed to MMC for 24 hr before staining for RAD51 foci. Foci were counted in GFP+ cells. Results show the mean of triplicate repeats from three independent experiments, ± SEM. Statistically significant pairwise comparisons are indicated (one-way ANOVA, followed by Dunnett’s post test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). See also Figure S4.
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7. Figure 5 IPMK Depletion Provokes Defects in Homologous Recombination
(A) Cal51 cells were depleted of IPMK(D2) and/or BRCA2 by RNAi, before exposure to the indicated concentrations of the poly-ADP ribose polymerase (PARP) inhibitor Olaparib. The percentage of viable cells was enumerated 5 days later, assigning 100% viability to siControl cells not treated with Olaparib. Results show the mean of triplicate repeats from two independent experiments, ± SEM. IC50 values (μM) for each treatment are shown.
(B and C) (B) Representative metaphases in BJ-5ta cells inducibly depleted of IPMK using (sh)RNAs exhibit chromosomal fusions and aberrant structures. shRNA expression was induced with 10 mg/ml doxycycline over 5 days. The histogram in (C) plots the frequency of cells with aberrant metaphases (%) with or without doxycycline treatment, before (blue columns) or after (red columns) exposure to MMC. A total of 172 metaphases were counted. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
An RNA Sequence Motif in the 3′ Untranslated Region of RAD51 Transcripts Mediates ALY Recognition
(A) Overrepresented motifs of lengths 6, 8, 10, and 20 nt from the untranslated and coding regions of transcripts downregulated in the cytoplasm of IPMK-depleted cells were determined using Multiple Em (expectation maximization) for Motif Elicitation (MEME). Two overrepresented 10-mer motifs in the 3′UTR of the transcripts identified are indicated.
(B) A biotinylated oligonucleotide encoding the consensus RNA motif GAGGCUGGGG but not CCCCAGCUCC retrieves ALY from nuclear extracts of Cal51 cells. Immunoblot analysis of the indicated proteins is shown.
(C) GST and GST-ALY expressed in E. coli and purified using glutathione Sepharose were analyzed by SDS-PAGE and Coomassie staining.
(D) RNA EMSAs using increasing amounts of purified GST or GST-ALY and a constant concentration of single-stranded GAGGCUGGGG RNA oligonucleotide demonstrate the dose-dependent formation of a GST-ALY:RNA complex. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
A Mechanism for IPMK Regulation of mRNA Export Selectivity through ALY
(A) Biotinylated RNA pull-down experiments were performed with equimolar amounts of the indicated sequences (full-length RAD51 3′UTR [1–980], mutant RAD51 3′UTR [1–660], and Ku70 3′UTR) using nuclear extracts from control siRNA-treated or IPMK-depleted cells, and immunoblotted for ALY.
(B) ALY and IPMK levels were monitored by immunoblotting with the indicated antibodies.
(C) Biotinylated RNA pull-down experiments were performed as above with equimolar amounts of Ku70 3′UTR or Ku70 3′UTR + GAGGCUGGGG, using nuclear extracts from control siRNA-treated or IPMK-depleted cells and immunoblotting for ALY.
(D) In vitro pull-down assays with IP3, IP4, and PIP3 beads and purified, recombinant ALY protein were revealed using Coomassie staining of the bound protein.
(E) Immunofluorescence of control siRNA-treated and IPMK-depleted Cal51 cells using anti-PIP2. Scale bar, 5 μm.
(F) Plots represent the average size of PIP2 foci calculated using ImageJ from at least 50 cells/experiment in three independent experiments, ± SEM.
(G) Biotinylated RNA pull-down experiments were performed with equimolar amounts of RAD51 3′UTR sequence using nuclear extracts from IPMK-depleted cells or identical extracts to which PIP3 (50 μM) had been added.
(H) A hypothetical model for the mechanism suggested by our findings is schematically illustrated. IPMK catalytic activity converts PIP2 to PIP3 in nuclear speckle domains (green oval). ALY recognizes target transcripts (blue) in the presence of PIP3, mediating their export from the nucleus to the cytoplasm through nuclear pore complexes (Nups). These include transcripts essential for DNA repair by HR, such as RAD51.
Molecular Cell  , DOI: ( /j.molcel )
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