Figure 4

RNA binding motif enrichment analysis indicates that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. (A) Shown is a comparison of gene expression levels of 89 known RNA binding proteins between hFK1 and hFK3 (see “Materials and methods”). (B) RNA binding motif enrichment analysis using rMAPS identifies four putative splicing factors: ESRP1, ESRP2 (see Fig. S5A), RBFOX2, and QKI. ESRP1 and ESRP2 have low expression levels in all Wilms’ tumor samples and monotonically increase along kidney development, starting with moderate levels in hFK1 and reaching a maximum in hFK3. Likewise, exons that were enhanced in hFK3 (with respect to hFK1) are enriched for ESRP1 binding sites at their downstream 3' flanking intron (red curve), while exons that are silenced in hFK3 are enriched for ESRP1 binding sites at their upstream 5′ flanking intron (blue curve). On the other hand, RBFOX2 shows a monotonic decrease in expression levels along kidney development, starting with high levels in hFK1 and decreasing in hFK2 and hFK3 (see discussion). Exons that are elevated in hFK1 (with respect to hFK3) are enriched for RBFOX2 binding sites at their downstream 3′ flanking intron (blue curve). Although QKI did not show a monotonic change in expression levels between hFK1, hFK2, and hFK3, its RNA binding sites show similar behavior to RBFOX2, that is, exons that are elevated in hFK1 (with respect to hFK3) are enriched for QKI binding sites at their downstream 3′ flanking intron (blue curve), and exons that are elevated in hFK3 (with respect to hFK1) are enriched for QKI binding sites at their upstream 5′ flanking intron (red curve). (C) These results are consistent with the model for splicing regulation during the Mesenchymal to Epithelial Transition (MET) as proposed by Yang et al.³¹. Applying this model to kidney development, the hFK1 cell fraction corresponds to early kidney developmental stages and is predominantly composed of mesenchymal cells (the cap mesenchyme and the un-induced metanephric mesenchyme). Therefore in hFK1, ESRP1 and ESRP2 are low and RBFOX2 and QKI promote exon inclusion by binding to downstream introns, or exon skipping by binding to upstream introns. The hKF3 fraction corresponds to a late more differentiated kidney developmental stage and is predominantly composed of epithelial cells. As a result, ESRP1 and ESRP2 are high in hFK3 and promote exon inclusion by binding to downstream introns, or exon skipping by binding to upstream introns.