Myosin Va Is Required for P Body but Not Stress Granule Formation

Abstract

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5-3 mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.Class V myosins are actin-based motor proteins composed of two identical heavy chains and several associated light chains. The amino-terminal head domain binds to ATP and actin filaments and provides the motive force for the complex. The neck region contains six calmodulin binding IQ motifs and acts as a lever arm, whereas the carboxyl-terminal tail domain mediates binding to cargo (1). Yeast have two class V myosins, flies and worms have one, whereas mammals have three (Va, Vb, and Vc). Myosin Va (MyoVa) 2 is widely expressed but is enriched in brain, testes, and skin. It has been implicated in a diverse range of cellular roles, including synaptic vesicle transport (2), insulin secretion (3), myelination (4), and long term potentiation (5). People who lack functional myosin Va suffer from a rare autosomal recessive disorder called Griscelli syndrome, which is characterized by hypopigmentation and severe neurological defects (6).It is well established that the yeast class V myosin Myo4p actively transports Ash1 mRNA to the bud tip of dividing cells (7), but it is now emerging that class V myosins from higher eukaryotes also play roles in RNA transport. Myosin Va has been found associated with several RNA binding proteins in a messenger ribonucleoprotein (mRNP) complex precipitated from a mouse brain extract (8). The intracellular distribution of RNA is dramatically altered in primary cells derived from dilute-lethal (myosin Va-null) mice (9). Yoshimura et al. (10) have shown that myosin Va mediates the translocation of TLS (translated in liposarcoma) and its target RNA, Nd1-L, into dendritic spines, and myosin V is involved in targeting oskar mRNA to the posterior po...

Processing-Body Movement in Arabidopsis Depends on an Interaction between Myosins and DECAPPING PROTEIN1

“…In animals and yeast, however, the actomyosin system has been implicated in the assembly and disassembly of P-bodies (Chang et al, 2008;Lindsay and McCaffrey, 2011). Human myosin Va colocalizes with P-body markers and is required for P-body integrity.…”

“…Actin, by contrast, is important for the delivery of RNA and P-body components to P-bodies and for their assembly. This is suggested by the colocalization of mammalian myosin Va with the P-body-associated protein eIF4E (for Eukaryotic Translation Initiation Factor4E) and further by the reduction of P-body number and transport of eIF4E to P-bodies when expressing dominant-negative myosin Va fragments (Lindsay and McCaffrey, 2011). An association of myosins with P-bodies was also reported in yeast.…”

Myrip Couples the Capture of Secretory Granules by the Actin-Rich Cell Cortex and Their Attachment to the Plasma Membrane

“…SiRNA duplexes were provided by MWG Biotech. We used three already validated siRNAs directed against human MyoVa (MyoVasiRNA1,5Ј-GAACAAAUGUGCACUCUUUUU;MyoVa-siRNA2,5Ј-AA AGUAAGGUCGUUGCUAAUU; MyoVa-siRNA3, 5Ј-AACUGACUAC CUGAAUGAUGA) (Desnos et al, 2007b;Lindsay and McCaffrey, 2011) and two siRNAs targeting Myrip (Myrip-siRNA1, 5Ј-UAAGUGAGCU GAAGCAGAAUU; Myrip-siRNA2, 5Ј-UGGCAGUGAUUCAACAUU U). The control siRNAs targeting EGFP or luciferase were from Eurofin MWG Operon.…”

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation

“…Initially identified through the use of an autoimmune serum targeting a glycine-tryptophan-rich protein, GW182, these membranefree structures are also called GW bodies (Eystathioy et al 2002). They are anchored to microtubules and move around in the cytoplasm (Aizer and Shav-Tal 2008;Lindsay and McCaffrey 2011). mRNA decay intermediates are found in P-bodies, and inhibition of 59-to-39 mRNA decay increases the size of P-bodies (Sheth and Parker 2003), suggesting that mRNAs can be degraded in P-bodies.…”