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The MUC1 Extracellular Domain Subunit Is Found in Nuclear Speckles and Associates with Spliceosomes

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      MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have dissimilar intranuclear distribution patterns.

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      Most cited references 82

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      Epithelia are protected from adverse conditions by a mucous barrier. The secreted and transmembrane mucins that constitute the mucous barrier are largely unrecognized as effectors of carcinogenesis. However, both types of mucins are intimately involved in inflammation and cancer. Moreover, diverse human malignancies overexpress transmembrane mucins to exploit their role in signalling cell growth and survival. Mucins have thus been identified as markers of adverse prognosis and as attractive therapeutic targets. Notably, the findings that certain transmembrane mucins induce transformation and promote tumour progression have provided the experimental basis for demonstrating that inhibitors of their function are effective as anti-tumour agents in preclinical models.
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        Pre-mRNA splicing: awash in a sea of proteins.

        What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.
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          A human cell line from a pleural effusion derived from a breast carcinoma.


            Author and article information

            Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, California, United States of America
            Northwestern University Feinberg School of Medicine, United States of America
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: PK GCD. Performed the experiments: PK LL TLT JWJ LM GCD. Analyzed the data: PK GCD. Wrote the paper: PK LL GCD.


            Current address: University of California Irvine, Irvine, California, United States of America

            Role: Editor
            PLoS One
            PLoS ONE
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            8 August 2012
            : 7
            : 8

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Pages: 18
            This work was supported by National Institutes of Health Grant 5R01HL068035 (GCD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Research Article
            Cell Membrane
            Membrane Proteins
            Protein Interactions
            Transmembrane Proteins
            Macromolecular Assemblies
            Molecular Cell Biology
            Cellular Structures
            Cell Nucleus
            Cellular Types
            Epithelial Cells
            Membranes and Sorting



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