Stefan Aigner

BACKGROUND: Haploinsufficiency of the RNA splicing regulator RBFOX2 has been linked to congenital heart disease, including hypoplastic left heart syndrome, yet its pathogenic mechanisms remain unresolved. METHODS: Human induced pluripotent stem cells heterozygous or null for RBFOX2 were created and their cardiomyocyte (CM) derivatives were characterized in monolayer culture and engineered heart tissues. In addition to cellular phenotyping, molecular profiling by RNA sequencing and enhanced crosslinking and immunoprecipitation sequencing was performed to detect changes in gene expression, exon usage, and RBFOX2 (RNA-binding fox-1 homolog 2) binding. These data sets were integrated to identify RBFOX2-bound RNAs with altered expression and splicing and to compare them with publicly available data sets from progressive CM differentiation time points. Rescue experiments were performed in induced pluripotent stem cell-derived CMs, engineered heart tissues, and zebrafish. RESULTS: We demonstrate that RBFOX2 is essential for CM differentiation by driving transcriptome maturation through gene expression- and alternative splicing-based isoform switching in transcripts primarily encoding sarcomere, cytoskeletal, and focal adhesion components, including the critical Z-disc factor ACTN2 (alpha-actinin-2). We further find that threshold levels of RBFOX2 facilitate autoregulatory splicing of mutually exclusive exons, generating early and late RBFOX2 isoforms with distinct functions. In heterozygous CMs, this threshold is not reached, resulting in isoform imbalance and production of an aberrant transcript, which further reduces functional RBFOX2 levels to well below 50% of wild-type. Finally, we demonstrate that ACTN2 overexpression rescues heterozygous, but not null, phenotypes, suggesting that ACTN2 activates an RBFOX2-dependent feedback loop that promotes transcriptome maturation. CONCLUSIONS: Our data demonstrate that reduced RBFOX2 dosage and impaired autoregulation stall CM differentiation by inhibiting transcriptome maturation, contributing to congenital heart disease pathogenesis and potentially increasing heart failure susceptibility following surgical intervention.

Supplemental Figure S2 shows validation of sgRNA -mediated knockout of hit RBPs, comparison of hit RBP depletion in RN2c in vitro versus in vivo, effects of ELAVL1 loss on human AML cell lines, characteristics of the MLL-AF9 AML mouse model, and ELAVL1 expression in normal hematopoietic cells.

Table S1 shows expression of RBPs in an RNA-sequencing data set of human bone marrow hematopoietic stem and progenitor subpopulations.

Table S2 shows sequences of (a) sgRNAs from the RBP screen, (b) sequencing primers, (c) shRNAs and human-targeting sgRNAs, (d) eCLIP oligos.

Table S9 shows the (a) differentially expressed transcripts and (b) altered pathways as identified by GSEA from the ELAVL1 knockdown human primary AML RNA-sequencing data set.

Supplemental Figure S4 shows the validation of ELAVL1 knockdown by western blot and effects of ELAVL1 loss on human primary AML cells in vitro and in vivo.

Supplemental Figure S5 shows the effects of DHTS treatment on THP-1 and human primary AML cells in vitro, the effects of MS-444 treatment on human umbilical cord blood in vivo, and validation of ELAVL1 overexpression by western blot.

Supplemental Figure S7 shows the expression profile of TOMM34 in normal human bone marrow and across different AML and CML data sets. The effects of TOMM34 depletion on human primary AML cells in vitro and validation of TOMM34 knockdown and overexpression is shown.

Supplemental Figure S3 shows the generation of the bcCML mouse model and the effects of ELAVL1 on normal bone marrow in vivo.

Table S5 shows the log2 fold change of differentially expressed transcripts from the ELAVL1 knockout RN2c RNA-sequencing data set.