The peripherin system of IFs in PC12 cells has provided a unique opportunity to observe the dynamic properties of a specific species of mRNA, along with its translation product. This is attributable to the finding that newly synthesized peripherin accumulates in close proximity to its mRNPs, which is demonstrated by the findings that ~30% of GFP-peripherin particles rapidly recover their fluorescence after photobleaching in a cycloheximide-sensitive fashion. This rapid recovery of fluorescence shows that the folding and maturation time for GFP-peripherin is on the order of minutes. Although the folding and maturation process of GFP takes ~1 h in bacteria and yeast (Rizzo and Piston, 2004
), it has recently been shown that GFP cDNA injected into the nuclei of mammalian cells is expressed and fluoresces in the cytoplasm within 40 min (Dean, 2004
). In addition, the refolding of extensively denatured GFP in vitro takes a few minutes, as determined by chromophore fluorescence (Enoki et al., 2004
). Furthermore, our FISH analyses demonstrated that a similar percentage (~29%) of peripherin particles was closely associated with peripherin mRNA and that this association was inhibited by puromycin. The loss of this association in the presence of puromycin shows that the relationship between peripherin mRNAs and protein particles depends on translation.
The interactions between peripherin particles and mRNA were directly visualized in living cells using the dual-transfection method developed for simultaneous imaging of both peripherin mRNA and its protein product. Time-lapse imaging shows active movement of both peripherin mRNPs and protein particles throughout the cytoplasm. However, the movements were independent of one another, and motile peripherin mRNPs were never seen associated with peripherin protein particles. Various mRNA species are known to move along both MT and microfilaments, powered by molecular motors such as kinesin, dynein, and myosin (for review see Tekotte and Davis, 2002
). Specific examples of kinesin-dependent mRNA movement include the mRNA-encoding myelin basic protein in oligodendrocytes (Carson et al., 1997
) and tau mRNA in axons of neuronal P19 cells (Aronov et al., 2002
). In addition, the localization of several zygotically transcribed mRNAs in Drosophila melanogaster
embryos have been shown to be dependent on cytoplasmic dynein (for review see Tekotte and Davis, 2002
). Although it has been hypothesized that motile mRNAs are not capable of translational activity, this has never been observed in vivo. Our dual peripherin mRNA- and protein-imaging method provides direct evidence that moving mRNPs are translationally inactive. In those instances where stationary peripherin mRNPs were found in close association with peripherin particles, the mRNP invariably moved rapidly away, leaving the protein particle behind. The reverse situation was never observed. In further support of these observations, the translational inhibitor puromycin increased the fraction of motile mRNPs to ~99%.
Visualizing the early stages of protein synthesis in association with peripherin mRNPs turned out to be extremely difficult because of the rapid motility of the majority of both mRNPs and protein particles along MT tracks. To observe the relatively small number of stationary mRNPs engaging in protein synthesis cells were treated with nocodazole. This disassembled MT and therefore stopped the movement of the vast majority of peripherin mRNPs not engaged in protein synthesis, as well as the particles not associated with mRNA. Under these conditions, we were able to observe peripherin particles forming in close association with those translation factories that had presumably formed before the disassembly of MT. In addition, ~70% of the mRNPs associated with particles were also associated with ribosomes. The ~30% that remained may represent mRNPs that have ceased the translational process and have already dissociated from ribosomes ( and Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200511033/DC1
). Most likely, newly synthesized peripherin particles will at some point engage molecular motors and initiate motility or alternatively, remain in place, ultimately assembling into short IFs that appear to link in tandem to form long IFs ( and Video 9; Helfand et al., 2004
). On the other hand, the departing mRNP may move to a new site of synthesis, associate with ribosomes, and begin another round of translation ( and Video 9).
Figure 8. A model depicting dynamic cotranslation mRNA particles. mRNPs (red) containing one or more peripherin mRNA are moved along MT (blue) by molecular motors such as kinesin and dynein. These motile mRNPs are translationally silent. When these complexes stop (more ...)
Quantitative analyses of peripherin mRNPs demonstrate that ~80% of total peripherin mRNA resides in clusters of two or more. Analyses of cells expressing the ECFP-TAA-Peripherin-R24-3′UTR construct, from which peripherin protein is not synthesized, demonstrate that ~60% of the total mRNA remains in clusters of two or more. The reduction from ~80 to ~60% is mostly attributable to an ~30% increase in mRNPs containing single mRNAs and an ~60% decrease in the largest mRNPs containing 6–30 mRNAs, suggesting that interactions between the nascent peripherin protein chains may contribute to the formation of large mRNA clusters. There was no significant change in the number of mRNPs containing two to five mRNAs under these experimental conditions. In the absence of a 3′UTR, there was a similar decrease in clustered peripherin mRNPs with the most dramatic decrease again in the large 6–30 mRNA-containing clusters. There was also an ~30% increase in mRNPs containing single mRNAs. The MS2-binding repeat region of the mRNA is most likely not involved in the mRNA clustering, as a previous work using the same repeat region conjugated to actin mRNA showed that the vast majority of the mRNA existed as single molecules (Shav-Tal et al., 2004
). Treatment of cells with nocodazole resulted in the most dramatic decrease in peripherin mRNA clustering, suggesting that the mRNAs are not clustered by simple diffusion, but require a regulated mechanism involving MT and most likely MT-based motors. The partial inhibition of clustering in nocodazole-treated cells may be because of an actin microfilament–based clustering mechanism (Stebbings, 2001
It has been suggested that the nonfilamentous peripherin particles contain dimers, which are the essential building blocks of IF, and perhaps higher order structures such as tetramers and ULFs (Chang and Goldman, 2004
). Dimer assembly requires the formation of coiled–coil interactions between the α-helical–rich central rod domains of two parallel and precisely in register IF protein chains (Strelkov et al., 2003
). The α-helical coiled–coil structure is one of the primary subunit oligomerization motifs in proteins, originally discovered in keratin IF by x-ray diffraction (McArthur, 1943
). The mechanism responsible for coiled–coil dimer assembly remains unknown. The finding that the majority of peripherin mRNA is located in clusters of two or more suggests that the formation of the coiled–coil interactions between pairs of peripherin protein chains takes place cotranslationally, involving the coordinated synthesis of closely associated mRNAs. Interestingly, during muscle differentiation, myosin assembly intermediates containing myosin heavy chain coiled–coil dimers also appear as globular “foci,” similar in appearance to peripherin particles. These foci are also thought to form cotranslationally (Srikakulam and Winkelmann, 2004
). The double FISH experiments showing the subpopulation of hetero-mRNPs containing both K8 and K18 mRNAs also support the possibility that the cotranslational assembly of IF dimers is a general phenomenon.
Our study provides evidence for the clustering of IF mRNAs and its physiological significance. Other mRNAs, such as β-actin, have been found to exist as single mRNAs, using the same quantitative FISH method that we have described (Fusco et al., 2004
). Furthermore, whereas peripherin mRNPs are distributed throughout the cell, actin mRNPs are concentrated in certain regions such as the lamellipodia of moving cells. This targeting of actin mRNPs is most likely related to the maintenance of high concentrations of monomeric soluble actin required for the extensive actin polymerization that takes place in lamellipodia (Shestakova et al., 2001
). In contrast, IF mRNPs can move to many sites distributed throughout the cell to initiate the synthesis and formation of insoluble, nonfilamentous precursors such as peripherin particles. This provides a cell with the capacity to control the de novo assembly of fully polymerized IF anywhere in its cytoplasm. This temporal and spatial control of IF polymerization may allow cells to locally regulate the distribution and various functions of IF, such as those involved in determining the mechanical properties of the cytoplasm and in signal transduction (Chang and Goldman, 2004
The direct observation of a specific mRNP and its protein product in the same living cell provides a powerful tool for determining the mechanisms and the order of events involved in targeted protein synthesis. Collectively, the results of this work reveal a process called dynamic cotranslation, which has profound implications for translational control in vertebrate cells.