Multiple functions for members of the myosin-V family have been characterized. Given the ancient origin of this family [Richards and Cavalier-Smith, 2005
; Foth et al., 2006
], this diversity of function is not surprising. These multiple functions make it more difficult to determine the molecular deficiencies responsible for the neurological and lethal phenotypes observed in homozygous Myo5a
null mutant mice and humans homozygous for null mutant MYO5A
alleles (Griscelli syndrome) [Pastural et al., 1997
Directional transport and localization of mRNA has been demonstrated to provide temporal and spatial control of gene expression [Huang and Richter, 2004
]. In neurons, the asymmetric distribution of mRNA contributes to synaptic plasticity [Burdwood, 1965
; Huang and Richter, 2004
; Huang et al., 2006
; Schuman et al., 2006
]. In yeast, it is well-established that a member of the myosin-V family, Myo4p, transports Ash-1
mRNA [Takizawa et al., 1997
; Beach and Bloom, 2001
; Muller et al., 2007
]. Therefore, an RNA transport/tethering deficit in Myo5a
null mutant mice may be sufficient to cause the physiological [Takagishi et al., 1996
; Miyata et al., 2000
] and morphological [Dekker-Ohno et al., 1996
; Takagishi et al., 2007
] phenotypes observed in the central nervous system. Circumstantial evidence suggesting a role for myosin-Va in RNA transport in vertebrate neurons was first published in 2002 [Ohashi et al., 2002
], but identification of a specific message associated with myosin-Va, Nd1-L, was only recently published [Yoshimura et al., 2006
We therefore set out to determine if myosin-Va was involved in RNA transport in a wider variety of cell types. Our results from quantitation of newly-synthesized RNA labeled by BrU after variation of pulse and chase intervals ( and ) suggest that myosin-Va functions as a transporter or tether at a very early stage of RNA transport, within 30 min of RNA synthesis. The prominence of differences in the vicinity of the nuclear membrane suggests that myosin-Va functions either inside the nucleus or just outside the nuclear membrane. There are preliminary reports of myosin-Va in the nucleus [Pranchevicius et al., 2008
] to support the former possibility; our future experiments will analyze isolated nuclei to distinguish between them. Our results suggest that another, albeit slower, mechanism exists for transporting RNA out of the nucleus, since newly-synthesized RNA merely leaves the nuclear region more slowly in the absence of myosin-Va.
Since null mutant pups grow more slowly than wild-type controls as soon as the phenotype is evident at 7–8 days of age, a trivial explanation for our observations is that the null mutant cells may not be as healthy as the wild-type cells. However, we observed no differences in total RNA content in PNS fibroblasts () or in distribution of RNA in spleen fibroblasts () between mutant and wild-type mice. The latter observation correlates with the low levels of Myo5a message and/or the low level of the brain-specific splicing pattern in spleen (). In combination with recent data demonstrating that interaction of dynein light chain 2 (DLC2) with the myosin-Va tail is dependent on the presence of the brain-specific exon B [Hodi et al., 2006
], our results contribute significantly to our understanding of the relevance of alternative splicing of the myosin-Va tail.
Mislocalization of β-actin mRNA in fibroblasts has been shown to change their direction of migration [Shestakova et al., 2001
], indicating that it plays a role in establishing cell polarity. Since it has been shown that β-actin mRNA distribution is dependent on actin filaments [Sundell and Singer, 1991
], we examined the distribution of β-actin message by in situ hybridization in wild-type and null mutant cells (). Wild-type cells had high concentrations of message in the periphery and just outside the nucleus, while the mutant cells had a more diffuse perinuclear distribution, with no increase at the periphery.
To assay for a physical association between β-actin message and myosin-Va, we immunoprecipitated myosin-Va and used RT-PCR to amplify β-actin mRNA from wild-type, but not null mutant, immunoprecipitates (). While this does not demonstrate a direct interaction, it is consistent with the in situ hybridization data. Our data are particularly interesting in the context of the demonstration that myosin-IIB has been shown in fibroblasts to be required for the proper localization of β-actin mRNA in response to growth factors [Latham et al., 1994
; Kislauskis et al., 1997
; Latham et al., 2001
]. From our pulse/chase experiments, myosin-Va appears to be responsible for mRNA transport from the nucleus to the periphery, where myosin-IIB could capture mRNAs.
These data are consistent with multiple models () that differ primarily in their sites of myosin-Va function. In the first model (), nuclear myosin-Va activity is required for the exit of some transcripts from the nucleus to areas from which the mRNAs can be transported anterogradely throughout the cell on microtubules. This model incorporates the preliminary observation of myosin-Va in the nucleus [Pranchevicius et al., 2008
], and fits well with the common involvement of microtubule-based motors in RNA transport [Kindler et al., 2005
], as well as the coincidence of myosin-V and kinesins on the same cargo [Huang et al., 1999
; Stafford et al., 2000
]. In mutant cells (), the absence of myosin-Va would slow exit of transcripts from the nucleus. The second model () incorporates a shuttle (or protective) factor that binds mRNA and reaches equilibrium by moving passively across the nuclear membrane. In mutant cells lacking myosin-Va, transcripts and the shuttle factor would fail to engage microtubule-based transport, and would accumulate in the perinuclear and nuclear regions, decreasing its rate of RNA exit from the nucleus. The granular perinuclear distribution of β-actin message in mutant cells () favors the second model (). Moreover, treatment of both mutant and wild-type cells with latrunculin A or nocodazole increases the nuclear/cytoplasmic ratio of BrU labeling (data not shown), consistent with both actin and microtubule involvement.
Fig. 7 Models for myosin-Va function in RNA transport in wild-type (A, C) and null Myo5a mutant (B, D) cells. (A, B) Nuclear myosin-Va functions in exit of newly-synthesized RNA (wavy lines) from the nucleus to the cytoplasm, also allowing for transport of the (more ...)
In summary, these observations parallel observations of myosin-Va function in melanocytes [Provance et al., 1996
], the loss of myosin-Va leads to a more granular and dispersed distribution of β-actin mRNA, and a greater nuclear/cytoplasmic ratio for both total and newly-synthesized RNA. Our pulse-chase experiments show that the impact of the absence of myosin-Va is only observed kinetically for newly-synthesized total RNA, but our in situ hybridization data show that myosin-Va has an essential function in β-actin mRNA distribution. We do not know if these functions are mechanistically identical. Any myosin-Va-dependent mechanisms for the proper localization of transcripts in fibroblasts are likely to share components with neuronal mechanisms for mRNA for transport, providing a significantly greater potential for experimental manipulation in fibroblasts.