Single mRNA dynamics in living cells
Fusco, Dahlene Nicole
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Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs, such as beta actin mRNA, are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs, or the poly-adenylated (poly(A)) mRNA population, are believed to be distributed throughout the cytoplasm. We have developed a method to visualize single mRNA molecules in living, non-polarized mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit periodic rapid and directional movements on microtubules. The beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a mechanistic basis for its localization activity. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are also involved in the other types of mRNA movements we have observed, which included complete immobility, restricted, and non-restricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.;We examined the mechanism of beta actin mRNA localization by reproducing the above analysis in primary, polarized cells. We described behavioral differences between localizing transcripts and non-localizing transcripts in polarized cells that included the sequence-dependent difference in movement type observed in non-polarized cells, as well as a unique packaging mechanism. We have observed the movement pattern of transcripts containing the beta actin 3' utr compared to transcripts with control (SV40) 3'utr and found that both transcript types exhibited directed movements, but the number of directed movements increased significantly in the presence of the beta actin 3'utr. Through single molecule studies of mRNA, we have determined that transcripts containing the beta actin zipcode were more likely to form packages, or aggregates of more than one molecule, than non-zipcode transcripts. Also, zipcode containing transcripts were more likely to combine packaging of mRNA molecules with directed movements. Packaging was not only sequence dependent but also serum-stimulation dependent. Unlike the sequence-dependent increase in directed movements, packaging did not occur in non-polarized Cos cells, providing evidence that it played a role in the mechanism of mRNA localization.;Finally, we have extended the analysis of mRNA particles at the single molecule level into the nucleus using cell lines containing stably integrated MS2 repeat loops. Through quantitative in situ hybridization analysis of these lines we have determined that the majority of mRNA particles in the nucleus contain single molecules when found at sites other than the transcription site and nuclear particle movements are most frequently diffusional.;Based on these results, we conclude that mRNA movement in the nucleus is significantly different from that in the cytoplasm, with greater frequency of directed movements in the latter compartment. Furthermore, all mRNA can undergo directed cytoplasmic movement and it is only the likelihood of such movement that is increased for localizing transcripts. Finally, mRNA that is capable of movement may be in the form of aggregates, or "granules", but single mRNA molecules are also capable of significant movement throughout the cytoplasm.