Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport.

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export.

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.

Close coupling between transcription and exit of mRNP from the cell nucleus.

Transcription is intimately coupled to co-transcriptional formation of mRNP particles and their preparation for export. In the dipteran Chironomus tentans we have now investigated whether on-going transcription is closely linked also to the ensuing transfer of the mRNPs from genes to cytoplasm. The assembly and nucleocytoplasmic transport of a specific mRNP particle, the Balbiani ring (BR) RNP granule, were visualized in larval salivary glands by electron microscopy. When transcription was inhibited with DRB or actinomycin D (AMD), the growing BR mRNPs disappeared from the genes. The two inhibitors affected the distribution of BR mRNPs in the nucleoplasm and in the nuclear pores in essentially the same way. At the nuclear pore complexes (NPCs) the basket-associated and translocating mRNPs were substantially reduced in number, the translocating RNPs being essentially absent after 90 min treatment. Remarkably, the amount of BR mRNPs in the nucleoplasm did not change. We conclude that on-going transcription is required for the mRNPs to exit from the cell nucleus. Interruption of transcription seems to primarily affect the intranuclear movement of BR mRNPs and/or prevent the binding of mRNPs to the NPCs rather than to directly interfere with translocation per se.