The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes

Priyadarsina Kumar, Louise Lindberg, Twanda L. Thirkill, Jennifer W. Ji, Lindsay Martsching, Gordon C Douglas

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numbere42712
JournalPLoS One
Volume7
Issue number8
DOIs
StatePublished - Aug 8 2012

Fingerprint

spliceosomes
Spliceosomes
Speckle
Small Interfering RNA
fractionation
Western blotting
epithelial cells
Western Blotting
Epithelial Cells
nuclear matrix
Staining and Labeling
Nuclear Matrix
Pancreatic Ribonuclease
Fractionation
trophoblast
antibodies
Antibodies
oncogenes
Trophoblasts
cell nucleolus

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes. / Kumar, Priyadarsina; Lindberg, Louise; Thirkill, Twanda L.; Ji, Jennifer W.; Martsching, Lindsay; Douglas, Gordon C.

In: PLoS One, Vol. 7, No. 8, e42712, 08.08.2012.

Research output: Contribution to journalArticle

Kumar, Priyadarsina ; Lindberg, Louise ; Thirkill, Twanda L. ; Ji, Jennifer W. ; Martsching, Lindsay ; Douglas, Gordon C. / The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes. In: PLoS One. 2012 ; Vol. 7, No. 8.
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