6 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

    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

    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

    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

    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

    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