背景:广泛表达的鸟嘌呤核苷酸交换因子,RAPGEF1(C3G),对小鼠胚胎的早期发育至关重要。它的功能是调节基因表达和细胞骨架重组,从而控制细胞增殖和分化。虽然已经预测了多个转录本,它们在小鼠组织中的表达尚未得到详细研究。
结果:全长RAPGEF1同工型主要是由于两个热点的剪接而产生的,一种涉及外显子3,另一种涉及外显子12-14,在蛋白质的Crk结合区之后立即掺入氨基酸。这些同种型在胚胎和成人器官中的表达不同。我们检测到未注释的存在,和不可预测的转录本,以各种组合的方式掺入盒外显子,特别是在心脏,大脑,睾丸和骨骼肌。同种型转换检测为培养中的肌细胞和小鼠胚胎干细胞分化形成肌管,和胚状体分别。盒式外显子编码富含丝氨酸的多肽链,本质上是无序的,并经历磷酸化。使用AlphaFold的计算机结构分析表明,盒外显子的存在改变了分子内相互作用,对于调节催化活性很重要。基于LZerD的对接研究预测,具有一个或多个盒外显子的同种型在与其靶GTP酶的相互作用方面有所不同,RAP1A.
结论:我们的结果证明了新的RAPGEF1亚型的表达,并预测盒外显子包含作为调节各种组织和分化过程中RAPGEF1活性的另一种手段。
BACKGROUND: The ubiquitously expressed Guanine nucleotide exchange factor, RAPGEF1 (C3G), is essential for early development of mouse embryos. It functions to regulate gene expression and cytoskeletal reorganization, thereby controlling cell proliferation and differentiation. While multiple transcripts have been predicted, their expression in mouse tissues has not been investigated in detail.
RESULTS: Full length RAPGEF1 isoforms primarily arise due to splicing at two hotspots, one involving exon-3, and the other involving exons 12-14 incorporating amino acids immediately following the Crk binding region of the protein. These isoforms vary in expression across embryonic and adult organs. We detected the presence of unannotated, and unpredicted transcripts with incorporation of cassette exons in various combinations, specifically in the heart, brain, testis and skeletal muscle. Isoform switching was detected as myocytes in culture and mouse embryonic stem cells were differentiated to form myotubes, and embryoid bodies respectively. The cassette exons encode a serine-rich polypeptide chain, which is intrinsically disordered, and undergoes phosphorylation. In silico structural analysis using AlphaFold indicated that the presence of cassette exons alters intra-molecular interactions, important for regulating catalytic activity. LZerD based docking studies predicted that the isoforms with one or more cassette exons differ in interaction with their target GTPase, RAP1A.
CONCLUSIONS: Our results demonstrate the expression of novel RAPGEF1 isoforms, and predict cassette exon inclusion as an additional means of regulating RAPGEF1 activity in various tissues and during differentiation.