53.3.1 scaRNAs Target U2 and U6 snRNAs and Both snRNAs Are Significantly Reduced in TOF
We used the scaRNABase database (http://www-scarna.biotoul.fr/index.php) to identify the nucleotides targeted for modification by the 12 scaRNAs that are reduced in TOF. Only two snRNAs were predicted to be targeted: U2 and U6 snRNAs. Six of the scaRNAs targeted 10 nucleotides (of 23 nucleotides that are known to be modified by scaRNAs) in U2 and 6 scaRNAs targeted 5 nucleotides (of 8 total modified nucleotides) in U6. Interestingly, U2 and U6 had significantly reduced expression in our 16 TOF samples compared to the 8 controls (U2 was reduced 1.8-fold in TOF RV, p = 0.04, and U6 was reduced 3.2-fold in TOF RV p < 0.0001). This is consistent with reduced stability of U2 and U6 as a consequence of inefficient scaRNA biochemical modification.
53.3.2 Cardiac Regulatory Networks Are Enriched for Alternative Splice Isoforms in TOF
Coordinated control of alternative splicing modifies the transcriptome and hence the proteome, while participating in most developmental processes [33, 34]. Studies of animal models have shown the importance of appropriate splicing for proper heart development [3, 5, 23, 35]. Still, knowledge of the sequence of events leading to tissue-specific alternative splicing is limited. More importantly with respect to the current proposal, nothing is known about the significance of scaRNA-guided nucleotide modification in spliceosomal RNAs and the potential impact on transcript splicing. Intriguingly, our analyses of splicing variants in TOF myocardium revealed a substantial increase of alternative transcript isoforms which were enriched in gene networks known to be critical for regulating heart development, including the WNT and NOTCH pathways. More importantly, ~50% of these alternative isoforms are present in normal fetal RV, suggesting regulation of splicing did not proceed properly during heart development in the infants with TOF (Alternative splicing of mRNA is dynamic in human fetal heart development, in prep). Figure 53.2 shows representative examples of alternative splicing, DICER and DAAM1, which have a similar pattern of splicing in fetal and TOF tissues (blue and green lines) compared to the control tissue (red line). Dicer was shown to be important in mouse heart development playing a role in splicing regulation , and splice variants of Daam1, a member of the WNT pathway, were recently shown to impact angiogenesis and endothelial cell migration and tube formation .
53.3.3 Reduced scaRNA Expression and Alternative Splicing Are Not a Consequence of Hypertrophy
Hypertrophy is known to reactivate the expression of some fetal genes. Thus we wanted to determine if the fetal expression patterns we observed could be a consequence of hypertrophy. As a comparison to TOF, we analyzed right ventricular samples from infants with TGA and PA/IVS. The embryological origins of TOF and TGA likely share some common features involving miscommunication between the first and second heart fields. However, PA/IVS is distinct and probably less complex in terms of origin of the regulatory defect. Nevertheless, TOF, TGA and PA/IVS share a common attribute: right ventricular hypertrophy. We observed only ten snoRNAs with differential expression in PA/IVS right ventricular tissue compared to control tissue. The ten snoRNAs that were reduced in PA/IVS were in common with TOF, and all target the 28S rRNA. In addition, U2 and U6 levels were not reduced in PA/IVS relative to the control tissue, and splice isoforms were essentially unchanged relative to the controls. On the other hand, RV from children with TGA bore a striking resemblance to the TOF pattern of scaRNA, spliceosomal RNA expression and fetal type splice isoforms. Therefore, the fetal type pattern of scaRNA and spliceosomal RNA expression, as well as fetal splice isoforms, does not appear to be simply a consequence of hypertrophy since they were not present in PA/IVS RV samples. These findings support our postulation that scaRNAs are of key importance in regulating heart development and not simply a consequence of hypertrophy.
53.3.4 Primary Cell Lines Derived from TOF Myocardium Retain the Same Relative Expression Patterns as the Tissue
We have derived primary cell lines from right ventricular myocardium obtained from 15 infants with TOF (TOF primary cells—TOFpc). These cells are most likely fibroblasts. The cell type is somewhat inconsequential since what we wish to examine is spliceosome response to changing levels of scaRNAs. We compared scaRNA levels, U2 and U6 levels, and splice isoform patterns between TOFpcs and primary myocytes derived from normally developing infant heart tissue. TOFpcs retained the same fetal type pattern of scaRNA, spliceosomal RNA expression and splicing isoforms of index genes relative to cells derived from normally developing neonatal cardiac tissue (data not shown). Splicing patterns also retained a TOF pattern in the TOFpcs relative to the normal cells. These changes were consistent through at least four passages of the cell lines.
53.3.5 Overexpression of ACA26 and SCARNA1 (ACA35) in TOF Primary Cells Was Associated with an Increase in U2 Levels and a Decrease in Fetal Splice Isoforms
The scaRNAs were cloned into an intron sequence between hemoglobin exons 3 and 4 so that they would be correctly processed in vivo and expression was driven with the CMV promoter . When either plasmid was transfected alone, there was an increase in the scaRNA but no change in U2 level or in splicing (data not shown). However, when we simultaneously transfected plasmids pCGL-ACA26 and pCGL-SCARNA1 into TOF primary myocytes, there was modest (~50%) but significant upregulation in U2 level. This is consistent with the idea that the scaRNA-directed modification of snRNAs is necessary for snRNA stability. More importantly, there was a reduction in the level of the fetal type splice form (Fig. 53.3). These data were repeated in TOFpcs from three different infants (genotypes). This is an exciting discovery which squarely supports our hypothesis that the scaRNAs are playing a role in spliceosomal function. These novel new findings support the critical role that scaRNAs play in mammalian heart development. This connection has not been investigated previously.
53.3.6 Knockdown of scaRNAs (scaRNA1 Targeting U2, or snord94 Targeting U6) Causes Heart Defects in Zebrafish
Studies of snoRNA-directed modification of ncRNA in archaea and lower eukaryotes have shown that nucleotide modifications are crucial for ncRNA functions [38, 39]. However, similar studies in vertebrates have not been described until the recent report of the developmental significance of snoRNAs in zebrafish by Kenmochi and colleagues . In summary, they suppressed the expression of several snoRNAs, including U26, in zebrafish (U26 was one of the snoRNAs that we identified as being down regulated in myocardium from infants with TOF). Using a unique highly sensitive mass spec analysis that they developed, they found that decreased U26 snoRNA expression reduced the snoRNA-guided methylation of the target nucleotides. Impaired rRNA modification, even at a single site, led to severe morphological defects and embryonic lethality in zebrafish. Thus, nucleotide modifications in rRNA play an essential role in vertebrate development. Additionally, as a preliminary study this past summer, in collaboration with Kenmochi and colleagues we have suppressed the expression of two scaRNAs, scarna1 and snord94 (both are homologous to scaRNAs identified by our screens of TOF myocardium) in zebrafish embryos. The knockdown of the scaRNAs resulted in developmental abnormalities, including heart malformations (Fig. 53.4) and altered splicing of genes that regulate heart development. This is an exciting finding and taken together with our observations that scaRNA levels impact splicing in TOF primary cardiomyocytes, suggesting that scaRNAs play a critical and, as yet, unrecognized role in vertebrate heart development. Furthermore, it appears that these particular scaRNAs primarily affect heart developmental processes. Collectively, these data provide firm support of our overarching hypothesis.
53.3.7 Splice Isoforms Change During Development in Zebrafish and After Targeted Knockdown of scaRNAs
We downloaded RNA-Seq data from the Gene Expression Omnibus derived from developing zebrafish at 0.75 h, 6 h, 1 day, 2 days, 3 days and 5 days postfertilization (GEO#: GSE30603) and analyzed for alternative splicing. We found clear changes in ratios of splice isoforms of a large proportion of genes, including genes important for heart development (e.g., Gata4, Mbnl1, Notch1, Dicer, data not shown). We performed RNA-Seq on RNA extracted from 24hpf zebrafish embryos treated with antisense morpholinos directed at scaRNA1 or snord94 and WT untreated embryos and embryos treated with mismatch morpholinos. Paired-end sequencing runs were performed with 101 base reads on the Illumina HiSeq 1500. RNA-Seq data were analyzed using the “Tuxedo suite.” There were no appreciable differences between mismatch morpholino and WT-type embryos. snord94 was reduced by 40%, and scaRNA1 was not detectable in their respective knockdown morphants compared to WT or mismatch treated embryos. We saw a clear shift in the predominant isoforms of our index genes after treatment with the antisense morpholino (Fig. 53.5 shows results of validation of the WNT pathway gene splicing variants in zebrafish morphants). These analyses clearly demonstrate that, as in mammals, splicing in developing zebrafish is dynamic. Furthermore, the knockdown experiments strongly support our hypothesis that scaRNAs are important for regulating splice variants and heart development. Furthermore, snord94 and scaRNA1 appear to be important for heart development.