PPIase dPPIL4 regulates high-amplitude PERIOD oscillations in Drosophila

Peptidyl-prolyl cis/trans isomerases (PPIases) accelerate proline peptide bond isomerization, affecting substrate protein function. In this study, through RNAi-based behavioral screening of PPIases in Drosophila melanogaster, we identified CG5808, termed Drosophila Peptidyl-Prolyl cis/trans Isomerase-like 4 (dPPIL4), as crucial for circadian rhythm regulation. Knockdown of dppil4 in clock cells lengthened the circadian rhythm period and decreased rhythmicity, accompanied by a significant reduction of core clock protein PERIOD (PER). Dppil4 knockdown downregulated per transcription and reduced phosphorylation at Ser5 in the RNA polymerase II C-terminal domain, critical for transcription elongation. In addition, dPPIL4 stabilized Cullin1 of the Skp1-Cullin1-F-box protein complex, a key regulator of PER degradation. Our findings suggest that dPPIL4 supports high-amplitude PER oscillation by enhancing both synthesis and degradation processes in a timely manner. In conclusion, our study underscores the importance of high-amplitude PER oscillations in PER for robust circadian rhythms and highlights the critical role of dPPIL4 in this process. Control flies and dppil4 knock-down flies (with knockdown in clock cells) were entrained under a 12:12 light-dark cycle. On the fourth day of entrainment, flies were collected at ZT8 and ZT16. Fly heads were isolated for RNA extraction, followed by RNA-sequencing.

肽基脯氨酰顺反异构酶（Peptidyl-prolyl cis/trans isomerases, PPIases）可催化脯氨酸肽键的顺反异构化，进而影响底物蛋白的功能。本研究通过对黑腹果蝇（Drosophila melanogaster）中的PPIases开展基于RNA干扰（RNAi）的行为学筛选，鉴定出基因CG5808——其被命名为果蝇肽基脯氨酰顺反异构酶样蛋白4（Drosophila Peptidyl-Prolyl cis/trans Isomerase-like 4, dPPIL4）——在昼夜节律调控中发挥关键作用。在生物钟细胞中敲低dppil4会延长昼夜节律周期并降低节律性，同时伴随核心生物钟蛋白PERIOD（PER）的表达量显著下调。敲低dppil4可下调per基因的转录水平，并降低RNA聚合酶II羧基末端结构域（RNA polymerase II C-terminal domain）上Ser5位点的磷酸化水平，而该磷酸化修饰对转录延伸至关重要。此外，dPPIL4可稳定Skp1-Cullin1-F-box蛋白复合物（Skp1-Cullin1-F-box protein complex）中的Cullin 1，而该复合物是调控PER蛋白降解的关键因子。本研究结果表明，dPPIL4通过适时增强蛋白质合成与降解过程，维持高振幅的PER振荡。综上，本研究证实了高振幅PER振荡对于维持稳健昼夜节律的重要性，并凸显了dPPIL4在该过程中的关键调控作用。将对照果蝇与生物钟细胞中dppil4敲低的果蝇置于12:12明暗周期中进行节律同步。在节律同步的第4天，于ZT8与ZT16时间点收集果蝇，分离头部组织以提取RNA，随后进行RNA测序（RNA-sequencing）。