Abstract
Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two-hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with β-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1-ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of β-actin mRNA but not of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant β-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced β-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.
Highlights
Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood
To probe mRNA transport in neurons, we looked at conventional kinesin and its subunits and cytoskeletal associations
Brief depolymerization of microtubules in primary mouse hippocampal neurons at 7 DIV significantly reduced ZBP1 along dendrites, interference with actin polymerization by latrunculin treatment had no apparent effect on the dendritic distribution of ZBP1 (Fig. 1, A and B; ***, p Ͻ 0.001, n ϭ 12 cells, 2 dendrites per cell)
Summary
Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with -actin mRNA in actively transported granules in living neurons. The aberrant -actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics These results suggest a critical role for PAT1 in BDNF-induced -actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates. KLC (Fig. S3) is involved in a mechanism for the fragile X mental retardation protein (FMRP)-dependent mRNA transport in neurons [11], which is significant because KLC is a bona fide cargo-binding subunit of kinesin-I [27, 28]. Distinct types of motor proteins have been shown to associate with mRNA cargoes, increasing the complexity of the mRNA-localizing mechanism [29, 30]
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