Huntington's disease protein contributes to RNA-mediated gene silencing through association with Argonaute and P bodies

Abstract

Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.microRNA ͉ poly-glutamine ͉ RNA interference ͉ post-transcriptional gene silencing ͉ neuronal RNA granule H untington's disease (HD) is caused by an expansion of the polymorphic stretch of uninterrupted CAG trinucleotide repeats in exon 1 of the IT15 gene, resulting in a long tract of polyglutamine (polyQ) in the N terminus of the HD protein huntingtin (Htt) (1). The human Htt protein contains 3,144 amino acids, and the polyQ stretch starts at the 18th amino acid, followed by a polyproline (polyP) sequence; the remaining portion of the protein is likely to be rich in ␣-helices, many of which compose HEAT repeats (2). The length of the glutamine repeats in the unaffected population varies from 6 to 35; HD is caused by expansion to 36 or more repeats (3, 4). HD is a member of a group of at least nine diseases caused by CAG repeat expansions that includes spinocerebellar ataxias (SCAs) 1-3, 6, 7, and 17, spinobulbar muscular atrophy, and dentatorubral-pallidoluysian atrophy. The protein that is subject to polyQ expansion in each disease appears to be unrelated, and despite their widespread expression in the brain and other tissues, mutation in each protein leads to distinct characteristic neurodegeneration patterns.Htt is expressed in a variety of tissues both within and outside of the nervous system. Many proteins have been demonstrated to interact with WT Htt and/or mutant Htt, and their functions support some of the mechanisms perturbed in HD, which include transcription, signaling, trafficking/endocytosis, and metabolism/mitochondrial functions (5-10). However, Htt's precise function has proven elusive, and our poor understanding of its function remains a limiting factor to the development of successful therapeutics (11). Proteolytic processing of WT and mutant Htt has been investigated extensively and appears to play a critical role in disease pathogenesis. Caspase-6 cleavage at amino acid 586 in mouse Htt and the release of the N-terminal Htt fragment are required for neuronal dysfunction and degeneration in an HD mouse model (12), but the basis for the need for this cleavage is unclear.We set out to search for binding proteins to elucidate the normal function of Htt. We chose a biochemical approach to purify proteins associated with WT and mutant Htt and ide...

Potential function for the Huntingtin protein as a scaffold for selective autophagy

“…These data support a role for HTT in ciliogenesis/ ciliophagy (52). A loss of WT HTT function may similarly cause dysregulation of RNA-mediated gene silencing because HTT associates with Argonaute and P bodies/stress granules, both shown to be cleared by selective autophagy/granulophagy (53)(54)(55). These associations suggest that many of the phenotypic consequences caused by loss of HTT function can be understood in terms of disrupted selective autophagy.…”

Proteomic Analysis of Wild-type and Mutant Huntingtin-associated Proteins in Mouse Brains Identifies Unique Interactions and Involvement in Protein Synthesis

“…Mutant Htt Purifications Implicate Altered RNA MetabolismWe have previously demonstrated that wild-type Htt functions with argonaute 2 (Ago2, the catalytic component of the RNAinduced silencing complex) in a post-transcriptional gene silencing pathway (14) and is also involved in RNA transport in cultured cortical neurons (48,49). The current study points to Htt function in protein synthesis through its interactions with ribosomal proteins, various translation initiation factors, and other accessory proteins.…”

“…Protein identification and quantification analysis were done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc. San Diego, CA) using ProLuCID, DTASelect2, and Census. Tandem mass spectra were extracted into ms1 and ms2 files (14) from raw files using RawExtract 1.9.9. Poor quality spectra were removed from the dataset using an automated spectral quality assessment algorithm (17).…”

Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis

“…20,21 Rhes, a striatal-specific guanine nucleotide-binding protein, interacts preferentially with polyQ-expanded Htt and induces its sumoylation, leading to cytotoxicity. 22 Interactors of Htt include several proteins related to RNA processing; 23,24 however, their relation to the polyQ expansion has not been elucidated. Two systematic studies of the Htt interactome have been reported to date: Wanker and coworkers used yeast two-hybrid screens to describe a protein interactions network for HD.…”

“…Mutant Htt associates with Ago2 in P bodies and contributes to RNA-mediated gene silencing. 23,24 P bodies, unlike SG, are not associated with ribosome and mostly serve for storage and degradation of repressed mRNAs. P bodies contain decapping enzymes and endonucleases and may associate with components of RISC complex (argonaute, DICER).…”