Mutant huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease [Ribo-seq]. Mutant huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease [Ribo-seq]

The polyglutamine expansion of huntingtin (mHtt) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis. Depletion of mHtt enhances protein synthesis and increases the speed of ribosome translocation, while mHtt directly inhibits protein synthesis in vitro. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicated a widespread shift in ribosome occupancy toward the 5’ and 3’ end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation by promoting ribosome stalling, a novel mechanistic defect that can be exploited for HD therapeutics. Overall design: For ribosome footprinting, three independent rounds of cultured stratial cells (i.e. control, HD-heterozygous, and HD-homozygous cells) were used. To resolve ribosome-associated footprints, the lysates were treated with RNase T1 and RNase A for 30 min, separated on 10-50% sucrose gradients and RNA from the 80S monosome fractions were purified.

亨廷顿蛋白（huntingtin, HTT）的多聚谷氨酰胺扩增突变体（mutant huntingtin, mHtt）可诱发亨廷顿舞蹈症（Huntington disease, HD）并引发神经退行性变，但其具体致病机制尚未阐明。本研究发现，突变型亨廷顿蛋白（mHtt）可促进核糖体停滞并抑制蛋白质合成。敲除mHtt能够增强整体蛋白质合成速率，加快核糖体移位速度；体外实验则证实mHtt可直接抑制蛋白质合成。我们进一步检测到核糖体蛋白与正在翻译的核糖体可与mHtt发生相互作用。通过高分辨率全基因组核糖体足迹测序（Ribo-Seq）与mRNA测序（mRNA-Seq）分析，研究团队发现与对照组相比，HD细胞内的核糖体占位谱整体向mRNA的5'端与3'端偏移，且在特定mRNA靶标上出现独特的单密码子暂停现象。综上，mHtt通过促进核糖体停滞阻碍翻译过程中的核糖体移位，这一全新的机制缺陷可作为HD治疗的潜在干预靶点。总体实验设计：针对核糖体足迹测序实验，本研究使用了三批独立培养的纹状体细胞（分别为对照组、HD杂合细胞与HD纯合细胞）。为分离核糖体相关的足迹片段，细胞裂解液经核糖核酸酶T1（RNase T1）与核糖核酸酶A（RNase A）处理30分钟后，通过10%-50%蔗糖梯度离心进行分离，随后纯化80S单核糖体组分中的RNA。