Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• An intronic GGGGCC repeat expansion in *C9orf72* is a leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• Sense and antisense transcription of these repeats produce dipeptide repeat proteins (DPRs), including poly(GA), poly(GR), and poly(PR), implicated in neurodegeneration.
• Poly(PR) interaction with RNA is hypothesized to contribute to its toxicity, but its transcriptome-wide binding profile remains uncharacterized.

Purpose of the Study:

• To identify the RNA targets of poly(PR) on a transcriptome-wide scale using crosslinking and immunoprecipitation (CLIP) analysis.
• To investigate the binding affinity and potential functional consequences of poly(PR)-RNA interactions, particularly concerning specific RNA motifs.

Main Methods:

• Crosslinking and immunoprecipitation (CLIP) in human cells to map poly(PR) RNA binding sites.
• Bioinformatic analysis to identify enriched sequences at binding sites.
• In vitro binding assays and phase separation experiments to assess poly(PR) interaction with specific RNA sequences.

Main Results:

• Poly(PR) was found to bind to nearly 600 distinct RNA molecules in human cells.
• The sequence motif GAAGA was significantly enriched at poly(PR) binding sites.
• In vitro studies demonstrated that poly(GAAGA) RNA exhibits higher binding affinity for poly(PR) and promotes its phase separation into condensates.

Conclusions:

• Poly(PR) exhibits preferential binding to RNAs containing GAAGA motifs.
• This specific RNA interaction may play a crucial role in the pathogenesis of C9orf72-associated neurodegenerative diseases.
• Further research into these poly(PR)-RNA interactions could reveal novel therapeutic targets.