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.神经元内的局部翻译，部分依赖停滞多聚核糖体（stalled polysomes）的再激活过程。然而，多聚核糖体发生停滞的具体机制仍未明确。停滞多聚核糖体可能富集于神经元RNA颗粒（RNA granules）中，这类颗粒以致密紧凑的核糖体集合为特征，可通过分离多聚核糖体与单核糖体（monosomes）所用的蔗糖梯度（sucrose gradients）离心得到的沉淀组分中被识别。我们从雌雄两性的出生后第5天（P5）大鼠大脑中分离得到该组分，发现其富含与停滞多聚核糖体功能相关的蛋白质，例如脆性X智力低下蛋白（fragile X mental retardation protein, FMRP）以及上游移码突变同源物1（Up-frameshift mutation 1 homolog, UPF1）。对该组分开展核糖体谱分析（ribosome profiling）显示，其核糖体足迹读数（footprint reads）大量来源于与神经元发育相关的细胞骨架蛋白的mRNA，同时在RNA结合蛋白的mRNA上呈现富集态势。此类足迹读数在3'端长于常规核糖体谱研究中的读数，且在mRNA的前半段区域呈现出具有重复性的峰形分布偏好。该类足迹读数富集于此前在体内与FMRP发生交联的mRNA所关联的基序中，这一结果独立证实了沉降沉淀组分中的核糖体与细胞内与FMRP结合的核糖体具有关联性。本研究数据支持如下模型：mRNA中的特定序列可介导神经元内翻译延伸过程停滞，进而招募FMRP，并启动停滞核糖体被包装并通过RNA颗粒进行转运的过程。