Movement and localization of RNA in the cell nucleus

The movement of various RNAs from their sites of chromosomal synthesis to t heir functional locations in the cell is an important step in eukaryotic ge ne readout, though one less well understood than the transcription, RNA pro cessing; 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 inve stigations my colleagues and I have undertaken over the past 7 years to des cribe the intracellular traffic and localization of RNA in living cells. On e approach we have developed is to glass-needle microinject similar to 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) hav e high affinity and has revealed very rapid movements of certain other RNAs from their nucleoplasmic injection sites to the nucleoli. One of these rap idly trafficking nucleolar RNAs is the signal recognition particle (SRP) RN A, and further results indicate that the nucleolus is a site of SRP RNA pro cessing or ribonucleoprotein assembly prior to export to the cytoplasm, In these fluorescent RNA microinjection studies, rye have also used mutant RNA molecules to identify specific nucleotide sequences that function as targe ting elements for the localization of RNAs at their respective intranuclear sites. In a second approach, we have used fluorescent correlation spectros copy (FCS), a classical biophysical method for measuring molecular motion i n vitro, coupled with confocal fluorescence microscopy to measure the. move ment 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 diffus ion in aqueous solution. Parallel experiments using the method of fluoresce nce recovery after photobleaching (FRAP) revealed a diffusion coefficient f or intranuclear poly(A) RNA close to that measured by FCS, These results be ar on the structure of the nucleoplasmic ground substance-an extremely cont roversial and unsolved problem in cell biology (29), The methods we have de veloped and these initial results represent the first major step toward a c omprehensive understanding of RNA traffic in the cell nucleus.