8 research outputs found

    REIIBP interacts with the SMN complex in myeloma t(4;14) cells.

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    <p>A) Identification of REIIBP binding partners. REIIBP protein partners were pulled down by a tandem affinity purification approach. To isolate a protein fraction with a low level of background contaminants, the lysates from H929::REIIBP and H929 (control sample) were passed through two different chromatography columns, an anti-Flag resin and Nickel resin. The purified populations were run on SDS acrylamide gel and stained with Coomassie blue. Discrete bands were cut from the gel and analysed by mass spectrometry. The number on the right indicates the position of the respective molecular weights in KDa. The positions for the proteins identified by Mass spectrometry are shown by arrows. B) Western blot analysis on Flag IP fractions. The names of the respective antibodies used for staining are shown. Molecular weight is shown. C–D) Endogenous REIIBP interacts with the SMN complex in myeloma cells. Co-IPs of GEMIN5 (C) and SMN (D) in t(4;14) untransduced myeloma cells. GEMIN5 and SMN were immuno precipitated from H929 cell lysates using specific antibodies. An IP with normal mouse IgG antibody was used as control. The IPs were resolved in a SDS acrylamide gel and blotted for the indicated antibodies. The black star shows the position for the heavy chains of the antibodies used in the Co-IP. Please note MMSET II in H929 cells has a shorter molecular weight than canonical 150 KDa.</p

    A Novel Functional Role for <i>MMSET</i> in RNA Processing Based on the Link Between the REIIBP Isoform and Its Interaction with the SMN Complex

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    <div><p>The chromosomal translocation t(4;14) deregulates <i>MMSET</i> (<i>WHSC1</i>/<i>NSD2</i>) expression and is a poor prognostic factor in multiple myeloma (MM). <i>MMSET</i> encodes two major protein isoforms. We have characterized the role of the shorter isoform (REIIBP) in myeloma cells and identified a clear and novel interaction of REIIBP with members of the SMN (survival of motor neuron) complex that directly affects the assembly of the spliceosomal ribonucleic particles. Using RNA-seq we show that REIIBP influences the RNA splicing pattern of the cell. This new discovery provides novel insights into the understanding of MM pathology, and potential new leads for therapeutic targeting.</p></div

    List of proteins identified as co-enriched with REIIBP by mass spectrometry.

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    <p>Proteins are ordered relative to the number of mass spectra assigned to peptide sequences unique to each protein. Only proteins with at least 10 fold enrichment in spectral counts relative to control are shown. Sequence coverage denotes the percentage of total amino acid sequence covered by the peptides detected. Asterisks denote known members of the SMN complex: GEMIN4, GEMIN5, GEMIN3 (ddx20), SMN and MEP50 (wdr77).</p

    Intron retention assay.

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    <p>Intron retention qRT-PCR results for a subset of genes present in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone-0099493-t002" target="_blank">Table 2</a>. Intron values for the indicated genes are normalized against the nearest exons and the βactin mRNA values. Data are shown as relative to HeLa values (blue dotted line). Each analysis is an average of at least three independent experiments. The error bars shown are the SEM, ** p value <0.005, * p value <0.05.</p

    Overexpression of REIIBP in HeLa cells does not lead to changes in the pattern of histone methylation and lacks a clear cellular phenotype.

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    <p>A) Top panel shows the structure of WHSC1 gene. The position of the different translocation events in MM is shown by slashed lines. Grey and black ovals indicate non-coding and coding exons respectively, dotted straight line between exons represent the introns. Splicing event for the canonical longest MMSET isoform is shown by joining lines between exons. Exons 4a and 11, shown by white triangles, are used only for alternative splicing and are not present in MMSET II and REIIBP isoforms. Transcription start sites and first translated codons (ATG) for MMSET II and REIIBP are indicated respectively by bent black arrows and open arrows. Genomic position for ACA11 is shown by a white arrow. Bottom panel shows the protein structure of the two major isoforms of the whcs1 gene. The major domains of the proteins are, PWWP (Pro-Trp-Trp-Pro motif) a chromatin associated domains, HMG high mobility group, PHD plant homeo domain, SET set domain with pre and post motifs. The position of the nucleolus exclusion signal and nucleus localization signal sequences are based on published observations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-Keats2" target="_blank">[15]</a> and prediction analysis software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-Brameier1" target="_blank">[58]</a>. B) IF and confocal analysis on HeLa::REIIBP and HeLA::ΔSET. REIIBP-Flag (green) localizes in the cytoplasm and nucleolus in HeLa cells overexpressing REIIBP-Flag. Single nucleolus zoomed in as inset in each frame. C) Western blot analysis on nuclear extracts. The antibody used is indicated on the left. D) Percentage of Apoptotic cells in the shown line. Error bars come from the SEM (standard error of the mean) of 3 different analysis. E) Percentage of cells at different stages of the cell cycle. Each data is the average of three analysis. * p value <0.05. F) WST-1 proliferation assay for the indicated cells. Error bars are the SEM of 5 different analysis. C–F) The control is the empty lentivector transduced into HeLa. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493.s001" target="_blank">Figure S1</a>.</p

    REIIBP SET domain controls spliceosomal snRNPs abundance.

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    <p>A) Cellular snRNPs abundance. snRNPs were immunoprecipitated with anti SmB/B′ antibody, snRNAs were purified, reverse transcribed and quantified by qPCR, (compared to the level of βactin mRNA present in the input). Each analysis is an average of three independent IPs. B) SMN complex components and SmB/B′ protein levels. Total cell lysates were immunoblotted against the relative antibodies. The black arrow indicates GEMIN3 specific bands. c) snRNAs expression analysis. qRT-PCR of total RNA. Each value is an average of three independent analysis. Figures A and C are expressed relative to control (HeLa cells, dotted line). The error bars are the SEM. ** p value <0.006, * p value <0.05.</p

    REIIBP controls snRNPs assembly and proliferation in myeloma cells.

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    <p>A) Cellular snRNPs abundance. snRNPs were immunoprecipitated and analysed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone-0099493-g003" target="_blank">Figure 3 A</a>. Each analysis is an average of three independent IPs. Data are shown as relative to H929 values (blue dotted line). The error bars shown are the SEM. B) WST-1 proliferation assay for the indicated cells. Results represent the average of 3 independent assays. A–B) * p value <0.05. C) A schematic representation of the function and position of the MMSET gene and its products. MMSET II controls the di methylation on Lysine36 of Histone3 (2MeH3K36) and the tri methylation of Lysine 27 on histone3 (3MeH3K27) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-MartinezGarcia1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-Kuo1" target="_blank">[14]</a>. Aca11 has been reported to be in the nucleolus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-Chu1" target="_blank">[5]</a>, whilst the SMN complex has both cytoplasm and nucleus localization. The SMN complex is found in the nucleus at level of Cajal bodies or in the gem bodies. REIIBP and the SMN complex are likely to interact in the cytoplasm. REIIBP catalytic activity, possibly on the SMN complex, affects the number of spliceosome snRNPs particles causing splicing defects. The schematic representation of the SMN complex model is based on a previously proposed model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone.0099493-Pellizzoni2" target="_blank">[21]</a>. The structures for REIIBP, ACA11, MMSET II and snRNPs are arbitrary.</p
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