Functional analysis of a novel small peptide gene in Drosophila pattern formation

小齿是果蝇幼虫上皮细胞的一种特殊的表皮结构，是由胚胎表皮细胞突出形成的。在以往的研究中，我们已经发现精米（pri）基因在水稻小晶粒的形成中起着重要作用。为了阐明果蝇花纹形成的分子基础，我们通过一系列的遗传实验对pri基因进行了表征。pri转录本不包含任何超过49aa的orf，但10个短orf中有5个是进化保守的。利用一系列pri转基因进行的报告基因分析表明，具有共同septamer motif LDPTGQ/TY的前4个orf（11,11,11和32aa）是独立于全长pri cDNA转基因的。当将任意四种orf中的任何一种引入pri突变体时，小齿表型完全恢复。另一方面，含有移框突变的对照转基因对表型没有任何影响。这些结果表明pri编码多个小肽，这些小肽在树突形成过程中具有冗余功能。通过延时成像分析，我们发现pri突变体在小突形成过程中存在明显的肌动蛋白动力学缺陷。在对照胚胎中，肌动蛋白纤维的细胞内定位预示着树突细胞中的细胞突起，而pri突变体没有显示任何定位信号的迹象。我们还发现，pri是转录因子Shavenbaby（SVB）控制的几种肌动蛋白调节因子表达所必需的，尽管SVB本身的表达不受pri突变的影响，这表明pri参与了SVB蛋白的转录调节。综上所述，我们得出结论，pri编码多种小肽，通过细胞间相互作用调节小突的形成。考虑到真核生物基因组包含许多短的ORF基因，我们的研究结果表明，小肽基因在发育事件中具有潜在的重要性。

Denticle is a special cuticle structure of larval epithelial cells in Drosophila, which is prepatterned by protrusion of embryonic epidermal cells. In previous studies, we have shown that the polished rice(pri) gene plays an important role in denticle formation. To elucidate molecular basis of pattern formation in Drosophila, we characterized the pri gene by a series of genetic experiments.pri transcript does not contains any ORFs longer than 49 aa, but five of ten short ORFs are evolutionarily conserved. Reporter assays using series of pri transgenes showed that the first four ORFs(11, 11, 11 and 32 aa), which share a common septamer motif LDPTGQ/TY, were independently translated from the full-length pri cDNA transgenes. When each of any four ORFs was introduced in pri mutants, the denticle phenotype was completely rescued. On the other hands, a control transgene containing frame-shift mutation did not show any effects on the phenotype. These results suggest that pri encode multiple small peptides polycistronically, which share redundant function during denticle formation.By time-lapse imaging analysis, we found that pri mutant shows evident defects of actin dynamics during denticle formation. In control embryos, intracellular localization of actin fibers prefigured cell protrusions in denticle cells, while pri mutants did not show any sign of the localized signal. We also found that pri is required for expression of several actin regulators controlled by transcriptional factor Shavenbaby(SVB), although expression of SVB itself is not affected by pri mutation, suggesting that pri is involved in transcriptional regulation by SVB protein.Taken together, we conclude that pri encodes multiple small peptides that regulate denticle formation via intercellular interaction. Taking account of the fact that eukaryotic genomes contain a number of short ORF genes, our results suggest that potent importance of small peptide genes in developmental events.