Arginine methylation of RNA-binding proteins is impaired in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD.

Evaluation of silica spin­column and magnetic bead formats for rapid DNA methylation analysis in clinical and point­of­care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. ß-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in ß-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.