Shank1 mRNA: Dendritic Transport by Kinesin and Translational Control by the 5′Untranslated Region

Abstract

Dendritic mRNA transport coupled with local regulation of translation enables neurons to selectively alter the protein composition of individual postsynaptic sites. We have analyzed dendritic localization of shank1 mRNAs; shank proteins (shank1-3) are scaffolding molecules of the postsynaptic density (PSD) of excitatory synapses, which are crucial for PSD assembly and the formation of dendritic spines. Live cell imaging demonstrates saltatory movements of shank1 mRNA containing granules along microtubules in both anterograde and retrograde directions. A population of brain messenger ribonucleoprotein particles (mRNPs) containing shank1 mRNAs associates with the cargo-binding domain of the motor protein KIF5C. Through expression of dominant negative proteins, we show that dendritic targeting of shank1 mRNA granules involves KIF5C and the KIF5-associated RNA-binding protein staufen1. While transport of shank1 mRNAs follows principles previously outlined for other dendritic transcripts, shank1 mRNAs are distinguished by their translational regulation. Translation is strongly inhibited by a GC-rich 5 untranslated region; in addition, internal ribosomal entry sites previously detected in other dendritic transcripts are absent in the shank1 mRNA. A concept emerges from our data in which dendritic transport of different mRNAs occurs collectively via a staufen1-and KIF5-dependent pathway, whereas their local translation is controlled individually by unique cis-acting elements.

BDNF-induced local protein synthesis and synaptic plasticity

“…The ultimate function of the trans-acting factors is to recognize cis-acting elements in the mRNAs and target the transcripts to distal dendrites. However, some of these RNAbinding proteins can also act as translation repressors (Huttelmaier et al, 2005;Jung et al, 2006;Napoli et al, 2008;Richter, 2007), linkers to motor proteins (Dictenberg et al, 2008;Falley et al, 2009) or even regulators of mRNA decay (Kim et al, 2005).…”

“…This study, together with the purification of KIF5-associated granules (Kanai et al, 2004), provided the best evidence for motor-based transport of RNA granules along the microtubule cytoskeleton. Another evidence for an interaction of trans-acting proteins and motor-based transport systems came from studies showing that the dendritic transport of Shank1 mRNA requires KIF5C and KIF5-associated protein Staufen1 (Falley et al, 2009). FMRP is also one of the best described factors that regulate the translation of mRNAs at the synapse.…”

Selective synaptic targeting of the excitatory and inhibitory presynaptic organizers, FGF22 and FGF7

“…To examine whether these KIFs are involved in FGF22 transport, we generated GFP-tagged dominant negative (DN) mutants for KIF5, KIF3A and KIF17, and short hairpin RNA (shRNA) for KIF21B (Labonté et al, 2013). DN-KIFs, which are also known as 'headless' mutants, contain the cargo-binding domain and no motor domain (Chu et al, 2006;Falley et al, 2009;Sathish et al, 2009;Uchida et al, 2009), and are mainly restricted to the cell body. The advantage of the headless approach is that they still bind to cargos and prevent compensatory transport by alternative systems.…”

“…Dominant negative (DN) forms of KIF5, KIF3A and KIF17 were designed to lack the motor domain but still bind to their cargos. The expression plasmid for KIF5DN was as described previously (Falley et al, 2009). Expression plasmids for KIF3ADN and KIF17DN were generated by subcloning a mutant form of human KIF3A or human KIF17 cDNA into the EcoRI-SalI sites of pAcGFP-C1 (KIF3ADN) or pAcGFP-C2 (KIF17DN) (Clontech).…”

Transport along the dendritic endoplasmic reticulum defines the trafficking modality for GABAB receptors

“…Kinesin-1 co-immunoprecipitates with GABA B1 in brain extracts (Vidal et al, 2007), participates in the axonal localization of GABA B1 (Valdés et al, 2012) and has been implicated in ER dynamics (Woźniak et al, 2009). However, the expression of a dominant-negative version of kinesin-1 comprising the cargo-binding domain of Kif5C that interferes with endogenous kinesin-cargo interactions (KIF5CDN-RFP, data not shown) (Falley et al, 2009) had no effect on the GABA B1 mobile fraction (control RFP, 4463%; KIF5CDN-RFP, 4368%; P50.872, Student's t-test) or recovery kinetics in dendrites (control RFP, 7164 s; KIF5CDN-RFP, 6963 s; P50.290, Student's t-test) (Fig. 5A,B).…”