Ribosome Traffic Jam: A Signal For Progressive Neurodegeneration?

Ribosomes, which are around 200,000 in a typical mammalian cell, need to move along mRNA (15,000–60,000/cell) to decode the information into proteins. Because ribosomes perform this monumental task, it has been suspected that ribosomal movement relies on evolutionarily sculpted molecular mechanisms. Ribosome stalling can trigger neurodegeneration[69].
However, what controls the ribosomal movement and underlying mechanisms in neurodegenerative disease is unclear.

Polysome profiling results showed Huntington disease cells had a bigger polysome (PS) to monosome ratio compared to control cells, but diminished protein synthesis. Ribosome run-off experiments with harringtonine indicated that ribosomes run more slowly in HD cells (Fig. A) and have diminished translation (protein synthesis) (Fig. B). These data indicated that ribosome stalling is an ideal mechanism to explain this phenomenon, as it strongly reflects diminished protein synthesis phenotype in HD. Additional experiments revealed that normal HTT is a physiological inhibitor of ribosome movement and inhibitor of protein synthesis, which is exacerbated by mHTT (Fig. C).
Next, we isolated mRNAs from the slowly translating PS in HD-homo and control striatal cells, using a harringtonine-based ribosome run-off assay (RRA) followed by mRNA-Seq (PS-RRA-mRNA-Seq). We found that there were ~1,157 targets that showed significantly high mRNA abundance in the PS of HD-homo cells (p < 0.05) compared to the control polysome. These data suggest that the translation pool of mRNAs is quite distinct in slowly moving ribosomes in HD.
Furthermore, by employing the high-resolution Ribo-Seq tool, we were able to demonstrate that there are widespread alterations of ribosome occupancy (ribosome-protected fragments, RPF), codon-specific pauses, and an altered center of ribosome density in HD cells compared to control cells. Intriguingly, global Ribo-Seq also revealed targets such as cGAS, a DNA sensor, which showed enhanced ribosome occupancy on exon1 (Fig. D). Thus, besides a global slowdown of translation, certain transcripts such as cGAS show enhanced protein synthesis in HD. We confirmed that cGAS is upregulated in the human HD striatum and promotes inflammatory and autophagy responses in cellular HD models.

Outstanding questions

Previous evidence supports translational deficits in HD, but the mechanisms are unknown. By employing elegant biochemical and molecular biology tools, our lab has demonstrated a new role of HTT and mHTT in controlling protein synthesis, involving ribosome stalling. As HTT and mHTT bind to ribosomes, we are interested in identifying their ribosomal interactors. We are particularly interested in identifying “pause-fixing factors,” molecular complexes on the mHTT mRNA, which show enhanced ribosome occupancy on the exon1 preceding CAG repeats. HTT consists of 28-32 HEAT repeats that span the entire protein. Many translation regulators, such as eEF3, eIF4Gs, p97DAP5, GCN1, and FRAP/mTOR, contain HEAT repeats, which interact with rRNA and ribosomal proteins of the small ribosomal subunit, and it is proposed that this plays a role in the translocation of aminoacyl-tRNA from the A site to the P site on the ribosome. We are interested in elucidating the potential role of HEAT repeats in HTT on ribosome stalling. We are particularly interested in intracellular differences in protein synthesis in heterozygous HD conditions. We test the hypothesis that, depending upon the localization and stoichiometry of HTT vs mHTT within the neurons, the translation is either up- or downregulated in a compartmentalized manner.