In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.
We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.
Huang
S
S
Cold Spring Harbor Laboratory, New York 11724.
Deerinck
T J
TJ
Ellisman
M H
MH
Spector
D L
DL
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D015530:Nuclear Matrix / Q000378:metabolism / Q000648:ultrastructure; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011061:Poly A / Q000032:analysis* / Q000378:metabolism*; D012313:RNA / Q000032:analysis* / Q000378:metabolism*; D012333:RNA, Messenger
D002843:Chromatin; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A; D012313:RNA