Nucleocytoplasmic transport of proteins and poly(A) plus RNA in reconstituted Tpr-less nuclei in living mammalian cells

Background: It is known that Tpr is a component of an intranuclear long filament which extends from the nuclear pore complex (NPC) into the nucleoplasm. Since the over-expression of the full-length of or some fragments of Tpr in living cells leads to the accumulation of poly(A)(+) RNA within the nuclei, it is generally thought that a relationship exists between Tpr and the nuclear export of mRNA in mammalian cells. In contrast, the nuclear export of poly(A)(+) RNA was not inhibited in a double deletion mutant of yeast Tpr homologues (M1p1p and M1p2p). Therefore, the precise function of Tpr remains unknown. Results: By microinjecting two types of polyclonal antibodies which are specific to Tpr into the cytoplasm of living mammalian interphase cells, we succeeded in reconstituting the Tpr-less nuclei. In the Tpr-less nuclei, the localization of the major components of the NPC, the nuclear import of SV40 T-NLS substrates and the nuclear export of HIV Rev NES-substrates were not affected. However poly(A)(+) RNA accumulated in the non-snRNP splicing factor SC35-positive clusters, which became larger in size and fewer in number, compared with normal nuclei. Conclusion: These results indicate that Tpr plays a critical role in the intranuclear dynamics of RNA pol II transcripts, including the processing, intranuclear transport and targeting, as well as their translocation through the NPC in mammalian cells.