The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.
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.
Priyadarsini
P
Kumar
Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, California, United States of America.
Priyadarsina
P
Kumar
Louise
L
Lindberg
Twanda L
TL
Thirkill
Jennifer W
JW
Ji
Lindsay
L
Martsching
Gordon C
GC
Douglas
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D019556:COS Cells; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis; D018396:Mucin-1 / Q000378:metabolism*; D015530:Nuclear Matrix / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D018396:Mucin-1; D034741:RNA, Small Interfering