Jeong Hyunjin, Son Suhyeon, Lee Gyunghee, Park Jae H, Yoo Siuk
Department of Life Sciences, Yeungnam University, Gyeongsan, Gyeongbuk 38541, Republic of Korea.
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.
G3 (Bethesda). 2025 May 8;15(5). doi: 10.1093/g3journal/jkaf055.
The scarecrow (scro) gene encodes a fly homolog of mammalian Nkx2.1, which is vital for early fly development and for optic lobe development. Previously, scro was reported to produce a circular RNA in addition to traditional mRNAs. In this study, we report 12 different scro circular RNAs, which are either mono or multiexonic forms. The most abundant ones are circScro(2) carrying the second exon (E2) only and bi-exonic circScro(3,4) having both the third (E3) and fourth exon (E4). Levels of circScro(2) show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circular RNAs in aged fly brains. In silico analysis of the introns flanking circular RNA exons predicts 2 pairs of intronic complementary sequences; 1 pair residing in introns 1 and 2 and the other in introns 2 and 4. The first pair was demonstrated to be essential for the circScro(2) production in cell-based assays; furthermore, deletion of the region including intronic complementary sequence components in the intron-2 reduces in vivo production of both circScro(2) and circScro(3,4) by 80%, indicating them to be essential for the biogenesis of the 2 circular RNAs. Besides the intronic complementary sequence, the intron regions immediately abutting exons seem to be responsible for a basal level of circular RNA formation. Moreover, ectopic intronic complementary sequence derived from the laccase2 locus is comparably effective in circScro production, buttressing the importance of the hairpin loop structure formed by intronic complementary sequence for the biogenesis of circular RNA. Last, overexpressed scro alters outcomes of both linear and circular RNAs from the endogenous scro locus, suggesting that Scro plays a direct or indirect role in regulating the expression levels of either or both forms.
稻草人（scro）基因编码哺乳动物Nkx2.1的果蝇同源物，其对果蝇早期发育和视叶发育至关重要。此前有报道称，scro除了产生传统的信使核糖核酸（mRNA）外，还会产生一种环状RNA。在本研究中，我们报告了12种不同的scro环状RNA，它们要么是单外显子形式，要么是多外显子形式。其中最丰富的是仅携带第二个外显子（E2）的circScro(2)，以及同时具有第三个外显子（E3）和第四个外显子（E4）的双外显子circScro(3,4)。circScro(2)的水平在成年果蝇头部呈现出年龄依赖性增加，这支持了环状RNA在老年果蝇大脑中高度积累的总体趋势。对环状RNA外显子侧翼内含子的计算机分析预测出2对内含子互补序列；一对位于内含子1和2中，另一对位于内含子2和4中。在基于细胞的实验中，第一对被证明对circScro(2)的产生至关重要；此外，删除内含子2中包含内含子互补序列成分的区域会使circScro(2)和circScro(3,4)的体内产生量减少80%，表明它们对这两种环状RNA的生物合成至关重要。除了内含子互补序列外，紧邻外显子的内含子区域似乎也负责环状RNA形成的基础水平。此外，源自漆酶2基因座的异位内含子互补序列在circScro产生中具有相当的效果，这支持了由内含子互补序列形成的发夹环结构对环状RNA生物合成的重要性。最后，过表达的scro改变了内源性scro基因座产生的线性和环状RNA的结果，表明Scro在调节其中一种或两种形式的表达水平中发挥直接或间接作用。
