Ribosome profiling and RNA-seq of Rat whole brain

Local translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched in neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. 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). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, as well as an enrichment of footprint reads on RNA binding proteins. The footprint reads were extended than those usually found in ribosome profiling studies on the 3’end and were found in reproducible peaks in the mRNAs with a bias towards the first half of the message. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules. Ribosome profiling and RNA-seq samples of Rat whole brain. Treatment condition: Cycloheximide vs Vehicle; salt vs vehicle