PDF(Pubmed)
Abstract：
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, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old 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 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with 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.
摘要：
神经元中的局部翻译部分是由停滞的多体的再激活介导的。停滞的多聚体可以在颗粒部分中富集，定义为用于从单体中分离多体的蔗糖梯度的颗粒。延伸核糖体如何在mRNA上可逆地停滞和不停滞的机制尚不清楚。在本研究中,我们使用免疫印迹表征颗粒部分中的核糖体，cryo-EM和核糖体分析。我们发现这个分数，从P5大鼠的两性大脑中分离出来，富含与停滞的多体功能有关的蛋白质，例如脆性X智力低下蛋白（FMRP）和上移码突变1同系物（UPF1）。该部分核糖体的Cryo-EM分析表明它们停滞了，主要是在混合状态。该部分的核糖体谱分析揭示了（i）与FMRP相互作用并与停滞的多聚体相关的mRNA的足迹读数的富集，(ii)源自与神经元发育有关的细胞骨架蛋白的mRNA的足迹读数的丰度和(iii)在编码RNA结合蛋白的mRNA上的核糖体占据增加。与核糖体分析研究中通常发现的相比，足迹读数更长,并定位到mRNA的可重复峰.这些峰富含先前与体内FMRP交联的mRNA相关的基序，独立地将颗粒部分中的核糖体连接到与细胞中的FMRP相关的核糖体。数据支持一个模型，其中mRNA中的特定序列在神经元的翻译延伸过程中起到使核糖体停滞的作用。意义陈述：神经元将mRNA发送到RNA颗粒中的突触，在给出适当的刺激之前，它们不会被翻译。在这里，我们表征了从蔗糖梯度获得的颗粒部分，并表明该部分中的多聚体在具有延伸的核糖体保护片段的翻译停滞的特定状态下停滞在共有序列上。这一发现极大地增加了我们对神经元如何使用专门机制来调节翻译的理解，并表明许多关于神经元翻译的研究可能需要重新评估，以包括在用于分离多核苷酸的蔗糖梯度颗粒中发现的大部分神经元多核苷酸。
