Abstract
Research in the last 10 years has led to improved understanding of the genetic regulation of vertebrate heart development, but despite this effort, approximately 70% of all congenital heart defects (CHDs) still have an unknown etiology. Alternative splicing of mRNA has been documented to play roles in normal and abnormal development. Dysregulated splicing of mRNA has been shown to cause heart defects in mice, however a link between mRNA splicing and CHDs has not yet been shown in humans. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF) relative to the RV of normally developing infants. Moreover, there was a significant decrease in the level of 12 scaRNAs (small cajal body associated RNAs) in the RV from infants with TOF. These small noncoding RNAs guide the biochemical modification of specific nucleotides in spliceosomal RNAs that are critical for spliceosomal function. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and alteration in mRNA splicing of several genes that regulate heart development. We modified the expression of sets of scaRNAs and consequentially documented distinctive mRNA splicing, accompanied by corresponding protein isoform changes suggesting a unique contribution by each scaRNA. Furthermore, we knocked down two homologous scaRNAs in zebrafish and saw a disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. These combined results provide compelling evidence that scaRNAs contribute to the regulation of cardiac development by fine-tuning the fidelity of the spliceosome that adjusts exon retention as cell differentiation occurs. Importantly, our findings are consistent with the concept that disruption of mRNA splicing patterns during early embryonic development disturbs normal signaling pathways, resulting in conotruncal misalignment and TOF.
Highlights
Congenital heart defects (CHDs) are the most common birth defects and represent a substantial health care burden even in countries with advanced health care systems [1]
~50% of these alternative isoforms are present in normal fetal right ventricle (RV), suggesting regulation of splicing did not proceed properly during heart development in the infants with tetralogy of Fallot (TOF) (Alternative splicing of mRNA is dynamic in human fetal heart development, in prep)
We examined the noncoding transcriptome in myocardial tissue from children with tetralogy of Fallot (TOF) and observed changes in mRNA splice isoforms of genes that are critical for regulating heart development [29]
Summary
Congenital heart defects (CHDs) are the most common birth defects and represent a substantial health care burden even in countries with advanced health care systems [1]. We hypothesize that tissue- specific control of the pattern of scaRNA expression could provide temporal and spatial specificity to the spliceosome, providing a ubiquitous mechanism for regulating splicing in the developing embryo. While the epigenetic role that scaRNAs play in maintaining spliceosomal integrity and precision is currently unexplored in terms of human health, we have collected substantial evidence that scaRNAs and splicing patterns are tissue-specific and play a critical role in vertebrate cardiac development. We have examined the impact on spliceosome function made by dysregulated scaRNAs using human primary cells derived from the right ventricle of infants with TOF and from normally developing infant heart tissue. We will carefully characterize the synergy between scaRNAs and assess the impact on spliceosome function (i.e., splicing) and vertebrate heart development. It is our contention that scaRNA function may serve as a checkpoint in transcriptional processing, regulating many aspects of development, including heart formation
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