Analysis Proposal
Analysis Question
Can we validate whether the RNase III endonuclease DICER1 processes GBA intron-derived dsRNA into siRNA species, and whether this processing is functionally relevant to GBA-associated neurodegeneration?
Datasets
Small RNA-seq from human brain tissue (BRAIN Initiative dataset)DICER1 CLIP-seq data from SH-SY5Y neuroblastoma cells (ENCODE dataset)Total RNA-seq from patient-derived iPSC neurons with GBA mutationsPublic small RNA-seq datasets from substantia nigra tissue (AMP-PD consortium)
Methods
Small RNA-seq alignment: map reads from control vs DICER1 knockdown neurons to the GBA intron reference sequences, quantifying intron-derived small RNAs (18-25 nt) and testing for significant reduction upon DICER1 lossDICER1 CLIP-seq motif analysis: extract peaks from ENCODE DICER1 CLIP data to identify direct binding events on GBA intron transcripts using HOMER motif discoveryIn vitro DICER1 cleavage assay: incubate in vitro transcribed GBA intron dsRNA with recombinant human DICER1, resolve products on denaturing PAGE, and validate by Northern blot using intron-specific probesAGO2 RIP-seq following DICER1 knockdown: immunoprecipitate AGO2-complexed small RNAs to determine if GBA intron-derived siRNAs are loaded into the RNA-induced silencing complex
Expected Outputs
- Quantitative evidence: significant accumulation of unprocessed GBA intron dsRNA and corresponding depletion of 21-23 nt siRNA species in DICER1 knockdown cells (supports relationship)
- Binding confirmation: enriched DICER1 crosslinking peaks within GBA intron regions with canonical DICER1 binding motifs (supports relationship)
- Cleavage products: appearance of 21-23 nt RNA bands derived from GBA intron dsRNA upon recombinant DICER1 incubation (supports relationship)
- Negative result: absence of DICER1 binding, no small RNA generation, and no effect on GBA intron RNA stability even upon complete DICER1 knockout (refutes relationship)