Reactivation of stalled polyribosomes in synaptic plasticity

Abstract

Some forms of synaptic plasticity require rapid, local activation of protein synthesis. Although this is thought to reflect recruitment of mRNAs to free ribosomes, this would limit the speed and magnitude of translational activation. Here we provide compelling in situ evidence supporting an alternative model in which synaptic mRNAs are transported as stably paused polyribosomes. Remarkably, we show that metabotropic glutamate receptor activation allows the synthesis of proteins that lead to a functional long-term depression phenotype even when translation initiation has been greatly reduced. Thus, neurons evolved a unique mechanism to swiftly translate synaptic mRNAs into functional protein upon synaptic signaling using stalled polyribosomes to bypass the ratelimiting step of translation initiation. Because dysregulated plasticity is implicated in neurodevelopmental and psychiatric disorders such as fragile X syndrome, this work uncovers a unique translational target for therapies.M ost studies of translational control focus on initiation, the process where mRNAs recruit ribosomes and catalyze the first step of translation (1). This highly regulated and normally rate-limiting step of translation is followed by elongation and termination, resulting in completed proteins. Although multiple ribosomes on a given mRNA (a polyribosome) imply active peptide synthesis, we and others identified neuronal RNA granulesmotile aggregates of nontranslating ribosomes (2, 3). These electron-dense bodies contain single copies of synaptic mRNAs that are translationally silenced during their transport from soma to synapse (1, 4).Many models assume that neuronally transported mRNAs are translationally paused before completion of the initiation step of translation during transport. An appropriate synaptic signal would then activate translation (initiation/elongation/termination) of the granule mRNA. However, it is not clear how many free ribosomal subunits are present at synapses to support translation initiation. Further, at a typical translation elongation rate of six amino acids per s (5, 6), synthesis of larger synaptic proteins (e.g., microtubule-associated protein 1b; MAP1b) would take over 5 min even if initiation were immediate. These two factors constrain the speed and magnitude of synaptic translation and, thus, plasticity. As some forms of synaptic plasticity require rapid (<10 min) and localized activation of protein synthesis, an alternative model is wanting (7-9).We have previously proposed the concept of a neuronal RNA granule as a stalled polyribosome (10, 11). Ribosomal stalling has been shown to occur in lysates from a mouse neuroblastoma cell line and in an in vitro rabbit reticulocyte lysate translation assay programmed with brain homogenate (12). Whether neuronal ribosome stalling occurs in vivo is uncertain. We hypothesized that neuronal RNA granules contain paused ribosomes with incomplete proteins initiated in the soma before their packaging and transport to dendrites, where translation can be rapidly and loca...

The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes

“…The functional implications of higher-order polysomes in the cellular context could be that they serve as paused complexes from which translation can start quickly again when required, allowing rapid, local activation of protein synthesis. An example of this idea was described in a recent publication 21 , suggesting that neurons translate synaptic mRNAs upon synaptic signalling using stalled polyribosomes to bypass the rate-limiting step of translation initiation. Finally, the formation of a continuous mRNA channel system could have important functional implications in that it could serve to protect the mRNA from degradation.…”

Synaptic control of mRNA translation by reversible assembly of XRN1 bodies

“…First, we monitored the local changes in the protein synthesis rate upon a short stimulation with 30 mM NMDA or 100 mM DHPG, which stimulates metabotropic receptors. We treated cultured hippocampal neurons with agonists as reported previously (Antar et al, 2004;Cougot et al, 2008;Park et al, 2008;Zeitelhofer et al, 2008;Baez et al, 2011;Tatavarty et al, 2012;Graber et al, 2013). We treated the neurons with tetrodotoxin (TTX), a drug that blocks voltagegated sodium channels and spontaneous activity, and then we exposed the cells to a 5-minute pulse of 30 mM NMDA or 100 mM DHPG (see Materials and Methods).…”

“…We treated the neurons with tetrodotoxin (TTX), a drug that blocks voltagegated sodium channels and spontaneous activity, and then we exposed the cells to a 5-minute pulse of 30 mM NMDA or 100 mM DHPG (see Materials and Methods). For in situ metabolic labeling of proteins, we used a recently described strategy termed fluorescent non-canonical amino acid tagging (FUNCAT) that has been used to monitor dendritic translation upon exposure to brain-derived neurotrophic factor (BDNF) (Dieterich et al, 2010) and, more recently, to show the reactivation of stalled polysomes upon treatment with DHPG (Graber et al, 2013). We labeled the proteins synthesized before the stimuli with L-azidohomoalanine (AHA) and the proteins synthesized during the 15 or 30 minutes after the stimulus with Lhomopropargylglycine (HPG) (Fig.…”

“…NMDAR stimulation enhanced SX-body formation and inhibited translation at spines and dendrites, whereas mGluR activation provoked the dissolution of the SX-bodies and stimulated local translation. We compared the behavior of the SX-bodies to that of S-foci and FMRP-positive granules, also involved in translational regulation at the synapse (Antar et al, 2004;Baez et al, 2011;Darnell et al, 2011;Graber et al, 2013). Similar to the SX-bodies, the S-foci and the FMRP-positive granules transiently dissolved upon DHPG, although with different kinetics.…”

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

“…Indeed, there are specific contexts in which active translation on polysomes becomes stalled 21,22 which the reader should be aware of. Determining whether or not transcripts are actively engaged with the translation machinery can be done by a) treating the sample with puromycin, which unlike EDTA, causes 'run-off' of ribosomes that are actively translocating across a mRNA, or b) treating the sample with homoharringtonine which inhibits translocation of only the first ribosome at the start codon, again causing 'run-off' of any downstream translocating ribosomes.…”