Kidney Week

Abstract: SA-PO582

MicroRNA Content in Cells Is Globally Disrupted by the Nephrotic Syndrome-Related Point Mutation in XPO5

Session Information

• Noncystic Mendelian Diseases
  November 04, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Genetic Diseases of the Kidney

• 802 Non-Cystic Mendelian Diseases

Authors

• Gur-Wahnon, Devorah, Hadassah - Hebrew University Medical Center, Jerusalem, Israel

Devorah Gur-Wahnon,

• Ben-Dov, Iddo Z., Hadassah - Hebrew University Medical Center, Jerusalem, Israel

Iddo Z. Ben-Dov,

Background

MicroRNA (miRNA) are small noncoding RNA that regulate gene expression. Mature miRNA are crucial for the development and homeostasis of podocytes. Maturation of miRNA entails a multistage process that involves several proteins. Recently, steroid-resistant nephrotic syndrome (SRNS) in a child was attributed to a point mutation in Exportin-5 (XPO5), a protein that exports miRNA precursors from the nucleus to the cytoplasm (Braun DA 2016). However, whether or not the mutation affects the miRNA-related function of Exportin-5 is not known. We hypothesized that the V552I mutation impedes miRNA maturation, and that the association with SRNS may shed light on the roles of specific miRNA in podocytes.

Methods

We transfected XPO5-knockout (XPO5^-/-) HCT116 cells with plasmids encoding either wt XPO5 or XPO5[V552I] and compared global miRNA content with non-rescued XPO5^-/- cells and parental cells via small RNA sequencing.

Results

miRNA quantification confirmed global depletion of mature miRNA in XPO5^-/- compared to parental cells. Transfection experiments showed partial rescue of miRNA content with wt XPO5. Transfection of a plasmid encoding XPO5 harboring the V552I mutation did not rescue miRNA maturation (Figure). Conversely, mapping of small RNA reads to mRNA and rRNA was relatively higher in XPO5^-/- cells and mutant XPO5-transfected XPO5^-/- cells compared to parental and wt XPO5-transfected XPO5^-/- cells.

Conclusion

Together with predictions of disrupting impact of the V552I mutation, our experiments show that the SRNS-related XPO5 mutation may cause global reduction of mature miRNA. Further experiments should pinpoint specific miRNA and miRNA targets responsible for the podocyte phenotype.

Figure: Log-normalized global miRNA counts in HCT116 cells, XPO5^-/- HCT116 cells, XPO5^-/- HCT116 cells transfected with wildtype XPO5-encoding plasmid and XPO5^-/- HCT116 cells transfected with mutant (V552I) XPO5-encoding plasmid

Funding

• Government Support - Non-U.S.