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GN–mPUM1 localized exclusively to the mitochondrial matrix, whereas three repeats of MTS were required for targeting mPUM2-GC; use of only one or two MTS sequences failed to localize mPUM2-GC to the mitochondrial matrix. Short linker amino acids (AAA) were used to connect the signal sequences to the probes. Other signal sequences may be used for targeting the probes to the nucleus or chloroplasts (Emanuelsson and von Heijne, 2001). When the signal sequence is used, it is then necessary to verify localization of the probes inside the cells.
2009. Live-cell imaging of viral RNA genomes using a Pumilio-based reporter. Plant J. 57:758770. 36 Volume 3 Current Protocols in Chemical Biology Tyagi, S. 2007. Splitting or stacking fluorescent proteins to visualize mRNA in living cells. Nat. Methods 4:391-392. Tyagi, S. R. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. Voytas, D. 2001. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 9. M. 2002. Modular recognition of RNA by a human pumilio-homology domain.
11. Continue to incubate for a total of 72 hr after the transfection. 12. Proceed to Basic Protocol 3.