Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA.
The myosin superfamily is made up of at least 35 classes of actin motors. Each member is characterized by the presence of an ~80 kDa MD (motor domain) that binds actin and ATP. This domain is usually followed by an α-helical neck containing one or more IQ motifs that bind light chains, usually calmodulin; the longer the neck, the larger the step size. Most myosins also have a C-terminal tail and some have an N-terminal extension. These regions are the most divergent and confer class-specific properties such as membrane binding or kinase activity. Myosins can be single- or double-headed motors depending on the presence or absence of a long coiled-coil region in the tail that allows the myosin to dimerize. Humans possess 39 myosin genes that can be placed into 12 classes and participate in a multitude of cellular processes such as muscle contraction, endocytosis, exocytosis, cell adhesion and motility, and cell division .
The class V myosins
The class V myosins are conserved from yeast to humans. Of the five myosin genes in yeast, two belong to class V (Myo2p and Myo4p). Drosophila has one myosin V gene and mammals have three. Myo2p transports secretory vesicles, Golgi elements, vacuoles and peroxisomes into the emerging bud in Saccharomyces cerevisiae, whereas Myo4p transports mRNAs to the bud tip. The Drosophila melanogaster class V myosin transports secretory vesicles at the rhabdomere and also pigment granules in photoreceptor cells in response to light. In mammals, myosin Vb has been implicated in the transport of a wide range of plasma membrane receptors to the cell surface from recycling endosomes. Interestingly, the myosin Vb transport pathway appears to be targeted by a number of viruses including RSV (respiratory syncitial virus) and HIV. Missense and nonsense mutations in the myosin Vb gene cause MVID (microvillus inclusion disease) characterized by an intractable life-threatening watery diarrhoea that occurs soon after birth. Patients with MVID lack microvilli on the surface of their enterocytes and instead possess microvilli in intracellular vacuoles, indicating that the disease is caused by impaired trafficking of apical and basolateral proteins. Relatively little is known about myosin Vc; however, it has been implicated in secretory granule transport in epithelial cells . Myosin Va is the best characterized mammalian class V myosin. It is widely expressed, but is enriched in the brain, testes and skin. It has several tissue-specific splice isoforms generated by the alternative splicing of several exons in a region of the central stalk domain where the coiled-coil structure is disrupted. A series of elegant studies has demonstrated that a melanocyte-specific isoform of myosin Va is recruited to melanosomes via an indirect interaction with Rab27A and facilitates the capture of these organelles at the actin-rich periphery of the melanocyte, a step required for the efficient transfer of melanin into neighbouring keratinocytes . In the brain, myosin Va has been implicated in the Ca2+-induced transport of glutamate receptors into the dendritic spines of neurons , and it has recently been shown to pull the endoplasmic reticulum into the spines of Purkinje neurons . Whereas the majority of work to date has focused on the role of myosin Va in the transport of membrane-bound cargoes, there is a growing body of evidence that implicates it in the transport of mRNA transcripts which are packaged in non-membranous mRNPs (messenger ribonucleoprotein particles).
Myosin Va is a large homodimer with an N-terminal MD followed by a neck domain containing six IQ motifs and a long coiled-coil domain in the tail that mediates dimerization. In vitro, it is a highly processive motor taking up to 50 steps before dissociating from actin. The long neck domain allows the molecule to take 36 nm steps, which is the same length as the half-helical repeat of an actin filament. A GTD (globular tail domain) at its C-terminus plays a role in cargo binding, key regulators of which appear to be the Rab GTPases . Several Rabs can bind directly, or indirectly, to different regions of myosin Va [7,8]. Calcium is a key regulator of myosin Va activation, and at submicromolar concentrations of calcium, myosin Va adopts a triangular inactive conformation in which the GTD folds over and binds to the MD. The ATPase activity of the folded motor is reduced and it binds only very weakly to actin [9,10]. An increase in the calcium concentration results in the opening of the molecule into its active extended conformation (Figure 1).
The myosin Va conformational switch
RNA transport and local protein translation
mRNA localization is an important way for the cell to control the expression of a protein. The directed localization of an mRNA transcript not only targets its encoded protein to the correct region of the cell, but also prevents its expression elsewhere. This is important for proteins such as MAPs (microtubule-associated proteins) like MAP2 and tau which will bind to microtubules anywhere within the cell, or for proteins that may be toxic to the cell if expressed at the wrong location, such as MBP (myelin basic protein) whose mRNA is transported to the processes of oligodendrocytes and severely compromises cell function if expressed elsewhere. In fibroblasts, β-actin mRNA is transported to the leading edge where it promotes local cytoskeleton assembly, cell polarization and cell motility. Local protein expression also provides a means for the cell to respond rapidly to a local requirement for a protein. A local stimulus can regulate protein translation at the site of stimulation, overcoming the need for a signal to be sent to the nucleus to initiate transcription, the export of the mRNA from the nucleus, its cytoplasmic translation and finally the transport of the protein to the location where it is required. This is important in dendritic spines during synaptic transmission, and defects in mRNA targeting can have deleterious effects on synaptic function and protein synthesis-dependent synaptic plasticity. Local protein synthesis is also more economical since a localized mRNA can be translated multiple times, which is more efficient than translating the mRNA elsewhere and individually transporting each protein to its site of action. RNA transport also allows gene expression to be controlled independently in different parts of the cell which is particularly important in large highly polarized cells such as neurons, which have hundreds of localized mRNAs .
mRNAs that are to be transported are packaged into mRNPs. These non-membranous complexes engage with motor proteins that direct their transport along the cytoskeleton. The composition of these mRNP complexes depends on cis-regulatory elements present on the mRNA that are recognized by specific trans-acting RNA-BPs (RNA-binding proteins). These RNA-BPs usually serve the dual function of controlling the targeting of the transcript and repressing its translation. The inactivation of the RNA-BP at its final destination results in the release of the mRNA and subsequent translational activation. Depending on the cell type, mRNAs are either derepressed once they reach their destination or maintained in a translationally repressed state until specific signals lead to their activation. An example of such an RNA-BP is IMP1/ZBP1 which binds to a 54 nt ‘zipcode’ sequence in the 3′-UTR (untranslated region) of the β-actin mRNA and directs its transport along microtubules into the lamellipodia of fibroblasts. Another example of an RNA-BP that acts as a translational repressor and co-ordinates the transport of a subset of mRNAs is FMRP (fragile X mental retardation protein) (see below).
Myosin Va and RNA transport
It has been known for over 16 years that yeast Myo4p transports the Ash1 mRNA along actin cables into budding daughter cells. Ash1 encodes an unstable transcriptional repressor protein which controls a mating-type switch by repressing the expression of HO endonuclease. Myo4p has since been found to transport 22 other transcripts, including some that encode membrane proteins, into the daughter cell. It is only more recently that a mammalian class V myosin has been implicated in mRNA transport. In 2002, Ohashi et al.  immunoprecipitated the RNA-BP Purα and observed a ~200 kDa protein in the immunoprecipitating complex. They identified this protein as myosin Va by MS. They also observed the RNA-BPs mStaufen and FMRP in the immunoprecipitates along with myosin Va. We have since followed up on this initial report and have confirmed the association of myosin Va with FMRP (A.J. Lindsay and M.W. McCaffrey, unpublished work). We demonstrate that the interaction with FMRP requires calcium, suggesting that myosin Va must be in its open active conformation, and that it is a protein–protein interaction since RNase A does not abolish their binding. In the absence of myosin Va, the motility of FMRP-positive granules is significantly reduced, and it localizes to several large perinuclear aggregates rather than displaying its normal punctate pattern distributed throughout the cell. However, we did not observe any effect of the loss of myosin Va on the motility or distribution of Purα or mStaufen, suggesting that it only plays a role in the transport of a subset of RNA-BPs and their cargo. We propose a model in which newly assembled FMRP–mRNP particles require myosin Va to associate with a kinesin. The kinesin then transports the complex along microtubules to the cell periphery where myosin Va serves to capture it on to the actin cytoskeleton and mediate its local delivery to the site of mRNA translation.
TLS (translated in liposarcoma) is an RNA-BP that binds the Nd1-L mRNA which encodes an actin-stabilizing protein. It translocates into dendritic spines upon mGluR5 (metabotropic glutamate receptor 5) activation and is essential for normal spine development. TLS associates with conventional kinesin and, in 2006, Yoshimura et al.  demonstrated that it also associates with myosin Va. They showed that knockdown of myosin Va, or overexpression of a dominant-negative mutant, inhibits the translocation of TLS into spines resulting in a ‘granular’ localization of TLS in the dendrites (reminiscent of the FMRP aggregates we observed in mouse melanoma cells lacking myosin Va). However, loss of myosin Va did not affect the translocation of mStaufen into spines. The authors proposed a model in which the long-range transport of TLS and ‘TLS-type’ RNA-BPs, and their target mRNAs, is mediated by a kinesin along microtubules and the short-range translocation into the actin-rich dendritic spines, where the target mRNAs are to be translated, is performed by myosin Va (Figure 2).
Proposed model for myosin Va-mediated mRNA transport into dendritic spines
Further evidence that myosin Va is involved in RNA transport includes a global comparison of the distribution of RNAs in primary cultures of cells taken from dilute-lethal (myosin Va-null) mice which revealed some striking differences with the RNA pattern found in primary cells from normal mice . In fibroblasts isolated from the adipose tissue of dilute mice, there appeared to be a much greater concentration of RNA in the nucleus and perinuclear region in comparison with control cells, suggesting that myosin Va plays a role in the transport of RNA species from the nucleus itself, or from immediately outside the nucleus. This phenotype could be reversed by expression of exogenous full-length myosin Va in the mutant cells. Interestingly, nuclear pools of myosin Va and myosin Vb have been observed [15,16], suggesting that there may be a role for these motor proteins in nucleocytoplasmic transport. β-Actin mRNA was found to be present in myosin Va immunoprecipitates and the transcript displayed a diffuse evenly distributed pattern in dilute fibroblasts rather than a concentration at the cell periphery as is the case in normal fibroblasts.
The single class V myosin found in Drosophila is involved in the transport of oskar mRNA to the posterior pole of the oocyte . Oskar is exclusively translated at the posterior pole and is responsible for posterior patterning and formation of germ cells and the abdomen in the resulting embryo. oskar mRNA is mislocalized in didum (myosin V-null) oocytes and in wild-type oocytes overexpressing the tail domain of myosin V. Drosophila myosin V can interact directly with the kinesin heavy chain and it can immunoprecipitate oskar mRNA . The authors of this study propose that long-range transport of the oskar mRNP through the cytoplasm is mediated by kinesin heavy chain and this is followed by kinesin-independent but myosin V-dependent capture and short-range translocation of oskar, along actin filaments, to the posterior cortex.
Myosin Va and P-bodies (processing bodies)
P-bodies are cytoplasmic foci present in all somatic cells that are involved in the storage and turnover of mRNA. Transcripts that enter P-bodies are either degraded or maintained in a translationally silenced state until they exit and re-engage in translation. P-bodies contain most of the enzymes required for mRNA degradation and are the sites of siRNA (small interfering RNA)- and miRNA (microRNA)-mediated gene silencing. They are dynamic structures, and both dynein and kinesin have been reported to regulate their dynamics . Myo2p in yeast has also been found to complex with a number of P-body components and P-body disassembly is delayed in a Myo2 mutant strain . We have found a pool of myosin Va that localizes to P-bodies in mammalian cells, and myosin Va depletion promotes their disassembly .
The majority of research carried out to date on the mammalian class V myosins has focused on their role in the transport of cargo that is packaged in membrane-bound organelles; however, it is becoming increasingly clear that myosin Va, at least, is also involved in the transport of mRNA which is packaged in non-membranous mRNPs. This is an evolutionarily conserved function of class V myosins as it has long been established to take place in yeast. It is unlikely that myosin Va interacts directly with mRNA transcripts, as it has no known RNA-binding motifs, but rather associates with them indirectly via interactions with a subset of RNA-BPs. Important future work will be to obtain a more comprehensive picture of the RNA-BPs that associate with myosin Va and where on myosin Va they interact, and if these sites overlap with known cargo/Rab-binding domains. It will also be interesting to determine the signals involved in directing a myosin Va molecule to an mRNP rather than a membrane-bound transport vesicle. It will be important to identify the specific mRNAs whose transport is under the control of myosin Va and whether these differ depending on the cell type. It is also unclear whether myosin Va can also redirect certain mRNAs to P-bodies for storage or degradation, and under what circumstances this takes place.
Rab GTPases and Their Interacting Proteins in Health and Disease: A Biochemical Society Focused Meeting held at University College Cork, Cork, Ireland, 11–13 June 2012. Organized and Edited by Mary McCaffrey (University College Cork, Ireland).
This work was supported by a Science Foundation Ireland Programme Grant [grant number 09/IN.1/B2629 (to M.M.C.)] and a HRB-Marie Curie Postdoctoral Research Fellowship [grant number MCPD/2009/6 (to A.J.L.)].