Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

Abstract

The regulators that stall ribosome translocation are poorly understood. We find that polyglutamine-expanded mutant Huntingtin (mHtt), the Huntington's disease (HD) causing protein, promotes ribosome stalling and physiologically suppresses protein synthesis. A comprehensive, genome-wide analysis of ribosome footprint profiling (Ribo-Seq) revealed widespread ribosome stalling on mRNA transcripts and a shift in the distribution of ribosomes toward the 5' end, with single-codon unique pauses on selected mRNAs in HD cells. In Ribo-Seq, we found fragile X mental retardation protein (FMRP), a known regulator of ribosome stalling, translationally upregulated and it coimmunoprecipitated with mHtt in HD cells and postmortem brain. Depletion of FMRP gene, Fmr1, however, did not affect the mHtt-mediated suppression of protein synthesis or ribosome stalling in HD cells. Consistent with this, heterozygous deletion of Fmr1 in Q175FDN-Het mouse model, Q175FDN-Het; Fmr1 +/-, showed no discernable phenotype, but a subtle deficit in motor skill learning. On the other hand, depletion of mHtt, which binds directly to ribosomes in an RNase-sensitive manner, enhanced global protein synthesis, increased ribosome translocation and decreased stalling. This mechanistic knowledge advances our understanding of the inhibitory role of mHtt in ribosome translocation and may lead to novel target(s) identification and therapeutic approaches that modulate ribosome stalling in HD. One Sentence Summary:Huntington's disease (HD) protein, mHtt, binds to ribosomes and affects their translocation and promotes stalling independent of the fragile X mental retardation protein.Main Text:

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

“…As we wrote this manuscript a study was published that suggested 335 Htt has a physiological function of binding to ribosomes and slowing the speed of ribosome 336 translocation of many target mRNAs [32]. Intriguingly, mutant Htt further slowed protein synthesis rates 337 [32]. These data suggest that Httex1 may be slowing translation in trans rather than in cis (through 338 nascent Httex1-driven stalling).…”

“…Our data here suggests that expression of Httex1, at wild-type or mutant polyQ lengths, does not stall 334 on the ribosome during translation. As we wrote this manuscript a study was published that suggested 335 Htt has a physiological function of binding to ribosomes and slowing the speed of ribosome 336 translocation of many target mRNAs [32]. Intriguingly, mutant Htt further slowed protein synthesis rates 337 [32].…”

cGAS, a DNA Sensor, Promotes Inflammatory Responses in Huntington Disease

“…For the first time, we applied high-resolution ribosome sequencing technology to genetically precise knock-in HD cell models: immortalized STHdh Q7/Q7 (control), STHdh Q7/Q111 (HD-het), and STHdh Q111/Q111 (HD-homo) striatal neuronal cells, derived from WT, Hdh Q7/Q111 , and Hdh Q111/Q111 mouse embryos (37). We successfully generated high-quality reads and found new roles for HTT as a physiological suppressor of translational elongation by regulating ribosome movements (38). In global ribosome profiling data, we found that cGAS showed a high ribosome occupancy (ribosome protected fragments (RPF)/mRNA) estimated by ribosome protected fragments (RPF) counts in exon 1 in HD-het and HD-homo cells, while there were fewer reads in control cells ( Fig.…”

“…1C, arrow) and 172 (CGT)) in HD cells. The RFP/mRNA analysis of other known DNA sensors such as Toll-like receptor 9 , which is mostly restricted to blood and immune cells (40), is not expressed in striatal cells (38). The absence in melanoma 2 (AIM2), another cytosolic DNA sensor (41), is also induced in HD cells, but to a much smaller extent compared to cGAS (the ratio of RFP/mRNA for cGAS is 14 whereas for AIM2 it is 2.2) ( Fig.…”

“…RNAase foot printing, generation of cDNA libraries from ribosome protected mRNAs, generation of cDNA libraries and sequencing, generation of mRNA-seq libraries, Ribo-Seq, RNA-seq quality control and mapping the reads to UCSC browser and ribosome pause analysis, were carried out as described in our previous work (38).…”