Abstract
Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5′ region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG+1 and AUG+214), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5′UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG+1), directing translation initiation to the second in frame start codon (AUG+214). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG+1, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5′UTR.
Highlights
Local protein synthesis is a cellular mechanism to generate an asymmetrical protein distribution in polarized cells
It has been suggested that activation of metabotropic glutamate receptors activates synaptic protein synthesis, and that this process is inhibited by the RNA binding fragile X mental retardation protein (FMRP; [4,5,6])
By analyzing the contribution of individual upstream open reading frames (uORFs) to translational control, we demonstrate that an unusual translation initiation site is required to maintain translation of the Shank1 mRNA at a basal level
Summary
Local protein synthesis is a cellular mechanism to generate an asymmetrical protein distribution in polarized cells. Neurons employ this mechanism to provide specific newly synthesized proteins to dendritic segments, spines or postsynaptic sites [1]. Local changes in protein composition can occur e.g. at recently activated synapses as a prerequisite to the so-called input specificity of synaptic plasticity [2]. This model requires that certain mRNAs are present in dendrites in significant amounts, and that these messages will only be translated upon the appropriate stimulus, while otherwise being translationally silent. It is clear that besides a number of trans-acting factors, cis-acting sequence elements must exist on dendritic mRNAs, which enable control by these cellular pathways
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