A number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear 'processing' events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events, in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation. A few studies have utilized nuclear transplantation/ microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis. Two considerations for RNA transport studies are discussed: 1) RNA transport in vitro relates to nuclear swelling, and colloidally-active agents (such as polyvinylpyrrolidone) should be employed to prevent nuclear swelling; and 2) contamination of nuclear preparations by nucleus-absorbed cytoplasmic RNA is substantial, and a detergent-rinse procedure is required to remove this contamination. In considering trafficking of RNP through nuclear pores, conceptual difficulties are obvious. Flux studies with exogenous tracers have indicated patent pore diameters of 90-120 Å. However, the RNP which cross the nuclear envelope (presumably through pores) are all at least 200 Å in diameter. Further, evidence is presented showing that nucleoplasmin-coated gold particles (of diameter 200 Å) pass through central channels in nuclear pores. Whereas previous models of RNP transport have enjoyed the luxury of ascribing deformability (local unfolding) to these RNP, this clearly is not possible for the coated gold particles. These considerations suggest that facilitated translocation of RNP occurs in some manner, and that the mechanisms governing this translocation are not subject to the constraints suffered by exogenous tracers in a 'rigid-channel' model of the nuclear pore.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Clinical Biochemistry
- Cell Biology