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MBE RNA editing based RNA seq

1. Experimental Purpose

  • Scientific Question: What are the direct RNA targets and precise binding sites of RNA-binding protein X in living cells?
  • Design Rationale: Fusion of RNA-binding protein (RBP) to an RNA editing enzyme (APOBEC) creates a chimeric protein that marks RNA binding sites with C-to-U conversions, detectable by RNA-seq without crosslinking artifacts
  • Follow-up Studies: Motif analysis of binding sites, functional validation of key targets, structural studies of RBP-RNA interactions, comparison with other RBP mapping techniques (CLIP-seq)

2. Model System

  • Primary System: Human cell line relevant to RBP function (e.g., neuronal cells for neuron-specific RBPs, cancer cells for cancer-relevant RBPs)
  • Rationale: Cell lines provide controlled expression of the RBP-APOBEC fusion, high RNA yield, and consistent experimental conditions
  • Alternatives:
  • Primary cells (pros: physiological relevance; cons: transfection challenges, variability)
  • Mouse models with inducible RBP-APOBEC (pros: in vivo context; cons: complex, expensive)
  • In vitro transcribed RNAs (pros: defined sequences; cons: lacks cellular context)
  • Ethical Considerations: Cell line authentication, appropriate biosafety practices, responsible use of genetic modification tools

3. Measurement Approach

  • Techniques:
  • Doxycycline-inducible expression of RBP-APOBEC fusion
  • RNA extraction with DNase treatment
  • Strand-specific RNA-seq with high depth
  • C-to-U editing site identification
  • Technical Replicates: Duplicate RNA-seq libraries
  • Potential Biases:
  • Expression level variations of RBP-APOBEC (use inducible system with titrated expression)
  • Off-target editing (include catalytically inactive APOBEC control)
  • RNA degradation artifacts (optimize RNA extraction protocol)
  • Sequence context preferences of APOBEC (account for in computational analysis)

4. Group Setting

  • Experimental Groups:
  • Test: Cells expressing RBP-APOBEC fusion
  • Control 1: Cells expressing catalytically inactive APOBEC fusion (deaminase mutant)
  • Control 2: Cells expressing APOBEC only (no RBP)
  • Control 3: Cells expressing GFP-APOBEC (non-specific RNA binding)
  • Control 4: Uninduced cells (baseline editing)
  • Controlled Variables: Induction time, expression level, RNA quality, sequencing depth
  • Biological Replicates: 3-4 independent inductions and RNA preparations
  • Modified Design: Include time-course of induction to capture kinetics of editing, or RBP mutants with altered RNA-binding specificity

5. Data Analysis & Presentation

  • Data Processing:
  • Read alignment to reference genome
  • C-to-U conversion identification
  • Background filtering using controls
  • Peak calling to identify significant binding sites
  • Motif discovery in binding regions
  • Statistical Analysis:
  • Statistical significance of editing sites above background
  • Enrichment analysis for RNA types and cellular compartments
  • Correlation with RBP expression levels
  • Comparison with known binding motifs or CLIP-seq data
  • Data Presentation:
  • Genome browser tracks showing editing sites
  • Metagene plots of binding distribution across transcript regions
  • Motif logos for identified binding sequences
  • Venn diagrams comparing targets with other datasets
  • Functional enrichment maps of target RNAs
  • Validation Methods:
  • RT-qPCR validation of editing at selected sites
  • CLIP-seq or RIP-seq for orthogonal validation
  • Functional assays for key targets (e.g., minigene reporters)
  • Mutagenesis of binding sites to confirm direct interaction
  • RNA structure probing to assess structural context of binding