Movement and localization of RNA in the cell nucleus

The movement of various RNAs from their sites of chromosomal synthesis to their functional locations in the cell is an important step in eukaryotic gene readout, though one less well understood than the transcription, RNA processing, and various functions of RNA. The segregation of the many classes of RNA out into to their appropriate sites in the cell is, from a physical chemical point of view, a remarkable phenomenon. This paper summarizes investigations my colleagues and I have undertaken over the past 7 years to describe the intracellular traffic and localization of RNA in living cells. One approach we have developed is to glass-needle microinject approximately 0.01 pl of fluorescent RNA solutions into the nucleus or cytoplasm of cultured mammalian cells. This 'fluorescent RNA cytochemistry' approach has resolved intranuclear sites ('speCkles') for which premessenger RNAs (pre-mRNA) have high affinity and has revealed very rapid movements of certain other RNAs from their nucleoplasmic injection sites to the nucleoli. One of these rapidly trafficking nucleolar RNAs is the signal recognition particle (SRP) RNA, and further results indicate that the nucleolus is a site of SRP RNA processing or ribonucleoprotein assembly prior to export to the cytoplasm. In these fluorescent RNA microinjection studies, we have also used mutant RNA molecules to identify specific nucleotide sequences that function as targeting elements for the localization of RNAs at their respective intranuclear sites. In a second approach, we have used fluorescent correlation spectroscopy (FCS), a classical biophysical method for measuring molecular motion in vitro, coupled with confocal fluorescence microscopy to measure the movement of poly(A) RNA in the nucleus, with the interesting finding that these RNAs appear to move about inside the nucleus at rates comparable to diffusion in aqueous solution. Parallel experiments using the method of fluorescence recovery after photobleaching (FRAP) revealed a diffusion coefficient for intranuclear poly(A) RNA close to that measured by FCS. These results bear on the structure of the nucleoplasmic ground substance-an extremely controversial and unsolved problem in cell biology (29). The methods we have developed and these initial results represent the first major step toward a comprehensive understanding of RNA traffic in the cell nucleus.