Nucleocytoplasmic transport of proteins and poly(A)+ 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 (Mlp1p and Mlp2p). 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.


CONCLUSIONS
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.
Shibata
Satoshi
S
Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Matsuoka
Yosuke
Y
Yoneda
Yoshihiro
Y
D000906:Antibodies / Q000276:immunology; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D034882:Lamins; D008845:Microinjections; D022022:Nuclear Pore / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors
D000906:Antibodies; D034882:Lamins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C073597:TPR protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors