TY - JOUR
T1 - Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association
AU - Anadolu, Mina N
AU - Sun, Jingyu
AU - Kailasam, Senthilkumar
AU - Chalkiadaki, Kleanthi
AU - Krimbacher, Konstanze
AU - Li, Jewel Ty
AU - Markova, Teodora
AU - Jafarnejad, Seyed Mehdi
AU - Lefebvre, Francois
AU - Ortega, Joaquin
AU - Gkogkas, Christos G
AU - Sossin, Wayne S
N1 - Copyright © 2023 the authors.
PY - 2023/2/27
Y1 - 2023/2/27
N2 - Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryo-EM and ribosome profiling. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (i) an enrichment for footprint reads of mRNAs that interact with FMRP and that are associated with stalled polysomes, (ii) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and (iii) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.Significance Statement:Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.
AB - Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryo-EM and ribosome profiling. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (i) an enrichment for footprint reads of mRNAs that interact with FMRP and that are associated with stalled polysomes, (ii) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and (iii) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.Significance Statement:Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.
U2 - 10.1523/JNEUROSCI.1002-22.2023
DO - 10.1523/JNEUROSCI.1002-22.2023
M3 - Article
C2 - 36849416
SN - 0270-6474
JO - The Journal of neuroscience : the official journal of the Society for Neuroscience
JF - The Journal of neuroscience : the official journal of the Society for Neuroscience
ER -