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. 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.