Kidney Week

Abstract: TH-PO0285

DDX54 Stabilizes APOL1 via Binding Alu dsRNA at 3'-UTR in Response to Innate Immune Signaling

Session Information

• Glomerular Diseases: Cell Biology
  October 22, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Huang, Huihui, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Huihui Huang, PhD

• Francey, Lauren J., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Lauren J. Francey, MSc

• Tattersfield, Calum, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Calum Tattersfield

• Kelly, Jessica, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Jessica Kelly, MS

• Pollak, Martin, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Martin Pollak, MD

• Friedman, David, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

David Friedman, MD

Background

Targeting APOL1 mRNA is a promising therapeutic strategy for APOL1-associated nephropathy. Our recent study demonstrated that an RNA-binding proteininteracts with the APOL1 Alu double-stranded RNA (dsRNA) structure within the 3′UTR to regulate APOL1 mRNA levels through the innate immune pathway. However, the broader repertoire of RNA-binding proteins involved in this regulatory mechanism remains unclear. Here, we employed an unbiased approach to identify proteins that bind to the APOL1 3′UTR dsRNA and potentially regulate APOL1 mRNA expression.

Methods

To find APOL1 mRNA binding proteins, we used the APEX2 proximity biotinylation labeling system. Six repeated MS2 RNA hairpin sequences were linked to an APOL1 3’UTR construct. APEX2 was linked to MCP, a protein with high affinity for MS2 sequences. When APEX2/MCP protein binds to APOL1/MS2 mRNA, the APEX2 peroxidase uses H2O2 to generate short-lived biotin phenoxyl radicals that label proteins within a 20 nm radius (i.e. APOL1 mRNA binding proteins).
APEX2/MCP was stably transfected (lentivirus) and APOL1/MS2 was transiently transfected into HEK293 cells. Proximity labeling was initiated by the addition of H₂O₂ and biotin-phenol. The labeled proteins were pulled down by streptavidin beads and quantified by mass spectrometry. Gene ontology enrichment analysis was performed to identify RNA-binding patterns. Binding of candidate APOL1 mRNA proteins was validated in human podocytes. The impact of binding proteins on APOL1 mRNA levels was tested by Q-PCR.

Results

In the APEX2-MCP labeling system, RIP data confirmed that APEX2/MCP protein bound to APOL1/MS2 RNA. Gene ontology enrichment analysis indicated that the APOL1 mRNA binding proteins were enriched for regulators of RNA metabolism. By far the most abundant protein binding to APOL1 mRNA was DDX54, an ATP-dependent RNA helicase. DDX54 was predicted by RBPmap to bind the Alu dsRNA regions of APOL1 3’-UTR. Under IFN treatment, DDX54 knockdown accelerated APOL1 mRNA decay after actinomycin D treatment.

Conclusion

The APEX2-MCP labeling system identified RNA-binding proteins that bind to APOL1 3’-UTR and regulate APOL1 mRNA expression. DDX54 binds and stabilizes APOL1 mRNA, making it a potential target for regulating APOL1 nephropathy.

Funding

• NIDDK Support