RNA transport is well characterized in several systems (Bor and Davis 2004). In polarized somatic cells and during early vertebrate development, endogenous RNAs are visualized as large granules (Barbarse et al. 1995; Ainger et al. 1997; Carson et al. 1998; Roock et al. 2000; Krichevsky and Kosik 2001) or particles (Sundell and Singer 1990; Ferrandon et al. 1994; Forristall et al. 1995; Kloc and Etkin 1995) that move at rates of up to 4 to 6 ^m/min via cytoskeleton-associated motors (Bassell and Singer 1997; Chartrand et al. 2001; Saxton 2001; Kloc et al. 2002; Tekotte and Davis 2002). RNA movement can be monitored in real time in living cells using a method based on a modification of the yeast three-hybrid system developed to detect RNA binding proteins (Bertrand et al. 1998; Takizawa and Vale 2000). In this system, two genes are expressed to monitor RNA transport. One gene encodes GFP fused to the MS2 coat protein, a high-affinity RNA binding protein from the single-
stranded MS2 RNA phage which recognizes a specific 19-nucleotide RNA stem loop. This reporter protein also contains a nuclear localization signal (NLS) to restrict it to the nucleus in the absence of any RNA target. A second gene transcribes a hybrid RNA containing prolamine RNA sequence and tandem repeated (6 x) MS2 RNA binding sites fused to a GUS reporter gene. When these two genes are expressed simultaneously, the MS2-GFP binds to one or more of the MS2 binding sites, which enables one to follow the movement of this RNA in real time by following the GFP fluorescence.
Hamada et al. (2003a) adapted this GFP two-gene monitoring system to follow prolamine RNA movement in developing rice endosperm and het-erologously in tobacco BY-2 cells. When the MS2-GFP fusion protein was expressed by itself in BY-2 cells, native fluorescence was observed predominantly within the nucleus. When coexpressed with GUS-MS2-prolamine RNA, GFP was localized to the periphery of the cell, the presence of the large vacuole in these cells apparently restricting GFP fluorescence to the thin cytoplasm adjacent to the cell wall (Hamada et al. 2003a). A similar pattern was also evident in developing rice endosperm cells when MS2-GFP fusion protein was expressed alone. However, an entirely different distribution pattern was observed when both genes were expressed. Numerous small particles ranging in size from 0.3 to 2 ^m in diameter were readily evident. Although many of the particles were stationary, several moved within the focal plane across the cell demonstrating RNA movement. These prolamine transport particles generally moved unidirectionally in a stop-and-go manner, with an estimated average velocity of 0.3-0.4 ^m/s and instantaneous velocities of up to 10 ^m/s (Hamada et al. 2003a). These characteristics are indicative of movement driven by a cytoskeleton-associated motor protein (Bassell et al. 1999; Jansen 1999, 2001; Tekotte and Davis 2002).
The dependence on the cytoskeleton for RNA movement was supported by the use of drugs that disrupt this cellular structure. Under optimal conditions, RNA movement particles were easily observed for up to 30 min. When treated with cytochalasin D and latrunculin B, which disrupt actin filaments, particle movement was rapidly suppressed with only Brownian movement noted. Movement was also arrested by 2,3-butanedione monoxime, an inhibitor of the ATPase activity of skeletal myosin (Ostap 2002). In contrast, the microtubule drug nocodazole had no significant effect on RNA movement (Hamada et al. 2003a). The results indicate that RNA particle movement is dependent on intact microfilaments.
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