Patil Prakash, Kibiryeva Nataliya, Uechi Tamayo, Marshall Jennifer, O'Brien James E, Artman Michael, Kenmochi Naoya, Bittel Douglas C
Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan.
Ward Family Heart Center, Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA.
Biochim Biophys Acta. 2015 Aug;1852(8):1619-29. doi: 10.1016/j.bbadis.2015.04.016. Epub 2015 Apr 23.
Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. 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). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.
可变剪接（Alternative splicing，AS）在调节哺乳动物心脏发育中起着重要作用，但剪接失调与先天性心脏病（Congenital heart defects，CHDs）之间的联系尚未得到证实。我们报道，超过50%与心脏发育相关的基因在法洛四联症（Tetralogy of Fallot，TOF）婴儿的右心室（Right ventricle，RV）中发生了可变剪接。此外，12种小 Cajal 体特异性RNA（small cajal body-specific RNAs，scaRNAs）的水平显著降低，这些scaRNAs指导剪接体RNA中特定核苷酸的生化修饰。我们试图确定scaRNA水平是否会影响可变剪接模式和心脏发育。我们使用来自TOF婴儿右心室的原代细胞，以显示scaRNA水平与几个调节心脏发育的基因（如GATA4、NOTCH2、DAAM1、DICER1、MBNL1和MBNL2）的剪接异构体之间存在直接联系。此外，我们使用反义吗啉代寡核苷酸敲低斑马鱼中两种scaRNAs（scarna1和snord94）的表达，发现心脏发育出现相应破坏，同时心脏调节基因的剪接异构体也发生改变。基于这些综合结果，我们推测剪接体RNA的scaRNA修饰有助于微调剪接体，以动态选择mRNA剪接异构体。我们的结果与早期胚胎发育过程中剪接模式的破坏一致，这导致第一和第二心脏场之间的通讯不足，从而导致圆锥动脉干排列不齐和TOF。我们的发现代表了一种确定先天性心脏畸形潜在机制的新范式。
