2023-07-05

Isoform-specific functions of RNA binding proteins

Presentation by Megan Schertzer

Alternative splicing regulates ~95% of multi-exon genes. It is regulated by (1) cis-acting pre-messenger RNA sequences, and (2) trans-acting RNA-binding proteins.

Project 1. Pooled RNA-IP approach to investigate variant effects on RBP binding and splicing.

Project 2. Isoform-specific functions using Cas13 knockdown and cDNA overexpression.

AS changes protein output to define cell-type or condition specific functions. Potential effects:

  • Protein localization
  • Nucleic acid binding
  • Co-factor binding

(RBPs show isoforms themselves.)

RBFOX2
well-established splicing factor with complex isoform structure. Alternative start sites, and 2 alternative junctions.

Cas13 RNA knockdown

Design sgRNA for junction level, knockdown and measure qPCR. High throughput, versatile (can target exons, alternative starts/ends and alternative 5’/3’ sites), targets endogeneous transcripts, doesn’t change underlying DNA locus. Disadvantages: some isoforms do not have fully unique seq for gRNA targeting, limited to studying isoforms expressed in cell type, feedback loop (?).

cDNA overexpression

  • Can express any isoform of interest
  • Can express isoforms at similar levels
  • cleaner experiments using FLAG tag to isolate isoforms
  • Disadvantage: not scalable.
  • Can help to study protein localization, RNA binding

How to summarize potential functional differences of RBP isoforms and prioritize isoforms for follow-up studies?

How to scale up the cDNA overexpression system?