The assembly- and processivity-mediating features of motile particles for asymmetric mRNA localization are not well understood. Previous studies on mRNA-transport in yeast comprised experiments with RNP complexes purified from cell extracts [102], used partial complexes lacking the RNA cargo [164] or assembled complexes without knowing the exact stoichiometric requirements [159]. Recent in vitro reconstitution experiments clarified the stoichiometric ratios within a SHE complex [145] and provided a basis to study mRNP assembly and motility under well-defined conditions.
Since reconstitution experiments suggested equimolar concentrations of myosin and RNA in the SHE complexes [145], Dr. Dennis Zimmermann (University of Chicago, USA) was instructed to perform single-particle motility assays considering these requirements. Assembled particles containing RNAs with one ASH1-E3 LE indeed moved processively along actin in vitro and had a mean run length of 2.13 ± 0.89 µm (Appendix Figure 5.2 A, E, F). This is roughly twice as much as observed by Sladewski and colleagues who reported run lengths between 0.9 and 1.4 µm for single LE RNAs [159]. However, considering the distances that have to be traveled in vivo (~2-4 µm, depending on the size of the bud cell [199]), our result suggests that a significant fraction of SHE particles are likely able to move the whole distance from the mother cell to the bud tip of the daughter cell in one continuous run.
TIRFM experiments further showed that particles containing single LE RNAs had a mean velocity of 0.74 ± 0.20 µm/s (Appendix Figure 5.2 A, E, F). This observation is in close proximity to results from previous studies. For instance in vitro actin gliding assays revealed velocities for Myo4p ranging between 0.45 and 0.65 µm/s [160] and localizing particles in vivo were reported to move with velocities of 0.55 µm/s [199], 0.20-0.44 µm/s [134], and 0.63 µm/s [216].
The relevance of cargo RNA for complex assembly and its impact on particle motility has been controversially discussed [165], [159], [164]. To directly address this issue we compared particles with varying complex compositions. The tested experimental set-ups lacked RNA and either involved complexes that were composed of wild type components or comprised particles that carried an RNA binding-deficient She2p (N36S, R63K) mutant. Particle velocities of both experiments
118 (0.65 ± 0.15 µm/s and 0.62 ± 0.20 µm/s, respectively) as well as their run lengths (2.48 ± 1.02 µm and 2.02 ± 0.69 µm, respectively) were comparable with the results of the wild type set-up (velocity: 0.74
± 0.20 µm/s, run length: 2.13 ± 0.89 µm) (Appendix Figure 5.2 E, F). Thus our findings pinpoint to an RNA-independent activation of particle motility in vitro and rather support a previous study from Krementsova and colleagues that suggested a negligible role for the RNA in motor activation [164].
Although the existence of RNA-free complexes in vivo is unlikely, these findings are valuable to deepen the mechanistic understanding of how a molecular motor is activated.
In vitro reconstitution experiments showed that ASH1-bound She2p associates with two She3p-Myo4p heterotrimers, which in turn assembles the SHE-core complex and induces She3p-Myo4p dimerization [145]. Since in our hands the cargo RNA is dispensable for processive movement, the necessity of the She2p-She3p interaction was challenged next. We found that particles reconstituted with the She3p-binding deficient mutant She2p ΔhE [139] were not able to move along actin filaments (Appendix Figure 5.2 F). This result confirmed that the protein-protein interaction between She2p and She3p is essential for proper SHE-complex assembly and its motility. Therefore I can conclude that it is rather the interplay between the RNA-binding proteins She2p and She3p that activates motility than the ASH1-mRNA cargo itself.
ASH1 mRNA has not just one LE, but in total four elements [71], [61]. In a recent study by our lab, a combination of dynamic light-scattering analysis and sucrose gradient centrifugation was used to show that an RNA construct with two LEs results in large particles and indicated multimerization of SHE-core complexes [145]. To assess the potential effects of multiple motors on particle processivity, RNA with two LEs was subjected to single-particle motility assays. Photo-bleaching experiments showed twice as many fluorescence-intensity populations compared to the single LE-RNA containing particles, thereby confirming particle multimerization (Appendix Figure 5.3 D, E). The mean run length of these large particles (1.93 ± 0.68 µm, Appendix Figure 5.2 D-F) was essentially unchanged from the run length of particles containing single LE RNAs (2.13 ± 0.89 µm, Appendix Figure 5.2 A, E, F). The same was true for the mean velocities of both particles (single complex: 0.54 µm/s ± 0.13 µm/s versus oligomerized complex: 0.74 ± 0.20 µm/s). This finding again provides evidence against an RNA-based activation mechanism and supports the notion that neither more LEs, nor more motor molecules increase processivity.
However, our findings are in contrast to results from single particle motility assays performed by Sladewski and colleagues [159]. They showed that increasing the number of LEs in one RNA also
119 slightly extended the mean run lengths of SHE particles (single LE RNAs: 0.9-1.3 µm versus multi LE RNAs: 1.1-1.5 µm). These values were obtained by using a strong substoichiometric ratio of RNA over myosin (0.035 nM RNA and 25 nM myosin) and increased further by raising RNA and myosin concentrations (10 nM RNA and 250 nM myosin) to 1.4 µm for single LE RNAs and to 2.8 µm for a multi LE RNA [159]. This dramatic deviation from the correct stoichiometric ratios, which included an excess of motor molecules, could be one reason for the observed discrepancy. Nevertheless further technical differences could also be responsible for these contradicting results. While we used single F-actin filaments from rabbit skeletal muscles and wild type She2p for complex assembly, Sladewski and coworkers used stabilized yeast actin-fascin-tropomyosin bundles in combination with She2p as quadruple cysteine mutant. In summary, RNAs with multiple LEs and prolonged run lengths are unlikely to be essential for in vivo localization, but might act as modulators for efficient localization of RNAs.
Interestingly, also in Drosophila assemblies of high-order complexes with several oskar mRNAs have been found [217]. Here the dense packaging of oskar-RNP particles is thought to mediate translational repression. This observation is to some extent reminiscent of the large SHE particles that form in vitro [145] and could serve similar purposes in the active yeast mRNA transport.
Although in yeast we do not see any influence of cargo RNA on motor activation or processivity, a contrary effect is observed in the case of dynein-dependent transport in the Drosophila embryo.
Here the dosage of LE-containing RNAs like hairy and the protein levels of BicD and Egl play an active role in regulating the initiation and maintenance of minus-end-directed mRNA transport [218], [219], [109].
Apart from ASH1-mRNA localization, She2p is involved in the transport of about 30 other transcripts into the daughter cell [111], [112], [113], [114]. In this respect the simultaneous transport of two different mRNAs (ASH1 and IST2) is sometimes accomplished by the same particle in vivo [199]. We could recapitulate this observation in our reconstitution experiments in vitro and showed that particles including both Cy3.5-labeled ASH1-E3 and ATTO488-labeled IST2 RNA moved actively along actin filaments (Appendix Figure 5.2 E, F). Since fluorophores were directly attached to the RNA, this experiment also served as direct proof that the reconstituted particles indeed transported RNA cargo.
120 In summary this study showed that run lengths of SHE complexes observed in vitro are compatible with the long-distance transport in vivo. The reconstituted particles offered similar motility properties, which were independent of the presence of RNA. This demonstrated that the RBP She2p and not its cargo activates motility. We further showed that SHE complexes have a defined size but multimerize into larger particles upon binding of RNAs with multiple LEs and are even able to bind different mRNAs.