title: TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells.
abstract: Phosphorothioate (PS) antisense oligonucleotides (ASOs) have been successfully developed as drugs to reduce the expression of disease-causing genes. PS-ASOs can be designed to induce degradation of complementary RNAs via the RNase H pathway and much is understood about that process. However, interactions of PS-ASOs with other cellular proteins are not well characterized. Here we report that in cells transfected with PS-ASOs, the chaperonin T-complex 1 (TCP1) proteins interact with PS-ASOs and enhance antisense activity. The TCP1-β subunit co-localizes with PS-ASOs in distinct nuclear structures, termed phosphorothioate bodies or PS-bodies. Upon Ras-related nuclear protein (RAN) depletion, cytoplasmic PS-body-like structures were observed and nuclear concentrations of PS-ASOs were reduced, suggesting that TCP1-β can interact with PS-ASOs in the cytoplasm and that the nuclear import of PS-ASOs is at least partially through the RAN-mediated pathway. Upon free uptake, PS-ASOs co-localize with TCP1 proteins in cytoplasmic foci related to endosomes/lysosomes. Together, our results indicate that the TCP1 complex binds oligonucleotides with TCP1-β subunit being a nuclear PS-body component and suggest that the TCP1 complex may facilitate PS-ASO uptake and/or release from the endocytosis pathway.
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA Lliang@isisph.com.
authors.forename: Xue-hai
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Shen
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Medicinal Chemistry, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA.
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., Carlsbad, CA 92010, USA.
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D022003:Cell Nucleus Structures / Q000737:chemistry*; D056404:Chaperonin Containing TCP-1 / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D054735:Phosphorothioate Oligonucleotides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000032:analysis; D014162:Transfection; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors
chemical_list: D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D021122:Protein Subunits; D056404:Chaperonin Containing TCP-1; D020931:ran GTP-Binding Protein