Identification of RNA-Binding Proteins Regulating the Alternative Splicing of TREM2 Exon 4

Background/Objectives: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Most AD cases are sporadic, with both environmental and genetic factors contributing to disease onset and progression. TREM2, an AD susceptibility gene, encodes a receptor protein expressed on the plasma membrane of microglia and macrophages. In addition to membrane-bound TREM2, soluble TREM2 (sTREM2) is also present in the brain. sTREM2 can be generated through two distinct mechanisms: proteolytic shedding of membrane-bound TREM2 and alternative splicing of TREM2 exon 4. Because the mechanisms regulating the latter pathway remain unclear, we investigated the molecular mechanisms underlying TREM2 exon 4 alternative splicing, with a particular focus on RNA-binding proteins (RBPs). Methods: We designed guide RNAs (gRNAs) targeting sequences within exon 4. The gRNAs and an exon 4 splicing reporter were transfected into HEK293-based cells with inducible dCasRx expression to identify regions containing cis-regulatory elements involved in exon 4 splicing. RBPs interacting with the candidate region were subsequently identified by RNA pulldown followed by LC–MS/MS analysis. Results: We identified regions within exon 4 containing putative cis-regulatory elements involved in its alternative splicing and found that CELF1, CELF2, and DDX41 interact with RNA containing one of these regions. Knockdown experiments indicated that these RBPs differentially regulate exon 4 splicing. Conclusions: Our findings suggest that TREM2 exon 4 alternative splicing is regulated by multiple cis-regulatory elements and RBPs, including CELF1, CELF2, and DDX41. Together with our previous identification of CELF1 and CELF2 as regulators of TREM2 exon 3 splicing, these findings suggest that CELF1 and CELF2 play complex roles in the regulation of TREM2 alternative splicing.