| Autophagy is a self-digestive process that intracellular material or pathogens targeted into lysosomes for degradation.It is a quality control mechanism that relies on the precise coordination of numerous ATGs(Autophagy-related protein),several complexes and organelles.Due to its evolutionary conservation,the diversity of autophagic substrates,the sophisticated mechanisms and the physiological or pathological significance,autophagy has quickly become a hot topic in the field of life science.Dysregulation of autophagy closely related to of human health and diseases,such as aging,obesity,neurodegenerative diseases and cancer.As a physiological self-eating process,autophagy was initially thought to be a non-selective response of cells under certain environmental stress.With the deepening of research,we now know that many cellular materials can be degraded by selective autophagy.According to the type of targeted cellular material,selective autophagy can be divided into different categories:mitophagy(mitochondria),reticulophagy(endoplasmic reticulum),ribophagy(ribosomes),aggrephagy(protein aggregates),xenophagy(pathogens)and so on.Mitophagy is an important form of selective autophagy,which plays an essential role in the maintenance of mitochondrial quality control.Depends on several of selective autophagy receptors(SARs),damaged,aging or supernumerary mitochondria specifically recognized and targeted to lysosome for mitophagic degradation,thereby maintain cellular homeostasis.More and more studies have shown that the disorder of mitophagy pathway is closely related to a variety of physiological or pathological processes,such as terminal differentiation of red blood cells,paternal mitochondrial degradation and neurodegenerative diseases.Although it has made significant progress in this intriguing cellular process,the precise mechanisms of mitophagy remain unclear.New physiological mitophagic induction,specific small molecule inhibitors,specific regulators(especially the mitophagy receptors)and its signal transduction mechanism need to be further explored.And the relationship between mitophagy and physiological or pathological process is becoming a hotspot with urgent attention.Thus,identification of novel regulators in this signaling pathways will contribute to understand the regulation mechanism of maintain mitochondrial homeostasis,and also will provide a new target and strategy for the disease associated with dysregulation of mitophagy.More and more studies have shown that the disorder of mitophagy signaling pathway is closely related to a variety of physiological and pathological processes that affect human health.Although great progress has been made in the field presently,much more need to be investigated further in physiological mitophagy induction,mitophagy inhibitor,specific factors and signal transduction mechanism involved in the process.In addition,the relationship between mitophagy and physiological or pathological process is becoming a hotspot with urgent attention in the field of autophagy research.Therefore,searching for novel key regulators of this signaling pathway will contribute to understand the molecular mechanisms that maintain mitochondrial homeostasis,and also will provide a new target and strategy for the treatment of diseases associated with dysregulation of mitophagy.In order to identify novel regulators of mitophagy,and to further investigate the regulation mechanism of mitophagy,we constructed a novel screening system for identification of mitophagy regulators.The system combined high-content screening with the recent mitophagy detection technology based on the unique fluorescent protein mtKeima(mitochondrial-targeted form of Keima).In the screening based on a siRNA library,we identified that PRPF8(pre-mRNA processing factor 8)plays an important role in the regulation of mitophagy.PRPF8 protein is a key component of the spliceosome and involved in messenger RNA processing.Until now,the role of splicing factors in mitophagy remains unclear.Using fluorescence microscopy and western blot,we conformed that the knockdown of PRPF8 significantly inhibits mitophagy induced by hypoxia and CCCP.To exclude the off-target effects of siRNA,we transfected siRNA-resistant wild-type(WT)PRPF8 expression plasmids on the rescue of mitophagy after PRPF8 siRNA knockdown in HeLa cells,and found that siRNA-resistant GFP-PRPF8 was able to restore the mitophagic activity.These results indicate that PRPF8 is indispensable for mitophagy in mammalian cells.In the experiments,we found that knockdown of PRPF8 on the inhibitory effect of hypoxia-induced mitophagy was significantly stronger than CCCP-induced mitophagy.Thus we further explored the mechanism of PRPF8 on mitophagy under hypoxia.We then detected the colocalization of mitochondria and lysosomes during mitophagy by immunofluorescence,and found that PRPF8 knockdown significantly inhibit their colocalization.Furthermore,we found that PRPF8 knockdown obviously inhibited the formation of LC3 puncta and mitophagosomes under hypoxia.We further explored the molecular mechanism that PRPF8 regulates mitophagy.Through cytoplasm and nuclear separation and immunofluorescence,our results showed that PRPF8 always localized in nucleus and did not colocalize with mitochondria during both basal and hypoxia conditions.These observations suggest that the function of PRPF8 in mitophagy is linked to its role in the spliceosome,which is required for pre-mRNA(precursor mRNA)splicing.We further evaluated whether there are defects in mitophagy-related genes expression following PRPF8 depletion during both basal and hypoxia conditions.We found that ULK1,which plays a crucial role in the initiation of mitophagosome formation,was obviously decreased following PRPF8 depletion under hypoxia.To further investigate how ULK1 expression is regulated by PRPF8,we assessed the pre-mRNA splicing of ULK1 gene in control and PRPF8 knockdown cells by reverse transcription-polymerase chain reaction(RT-PCR).We found that knockdown of PRPF8 in HeLa cells led to enhanced ULK1 exon 22 and exon 22-23 skipping.We further found that the overexpression of GFP-ULK1 in PRPF8 knockdown cells also rescued the mitophagic activity under hypoxia.Thus,these results collectively suggest that PRPF8 is required for mitophagy by regulating ULK1 mRNA splicing and expression.Many mutations in the C-terminal appendix of human PRPF8 lead to progressive degeneration of the retina and RP13 finally,a severe form of adRP(autosomal dominant retinitis pigmentosa)in humans.We asked whether PRPF8 retinitis pigmentosa disease-associated mutants PRPF8-R2310K have any defects in regulating hypoxic mitophagy.Unlike the WT PRPF8,the R2310K PRPF8 was incapable of rescuing the mitophagy defect in PRPF8 knockdown HeLa cells.These data suggested that adRP associated PRPF8 R2310K mutation may be defective in regulating hypoxic mitophagy.Moreover,we observed that knockdown of BRR2,PRPF6 or PRPF31,which are mutated in adRP,also inhibit hypoxia-induced mitophagy.While knockdown of PRPF19,which are not mutated in adRP,have no effect on hypoxia-induced mitophagic activity.Furthermore,the knockdown of BRR2,PRPF6 or PRPF31 but not PRPF19 remarkably increased ULK1 exon 22 and exon 22-23 skipping and reduced the hypoxia-induced ULK1 expression.Thus,these results suggested that mitophagy and ULK1 splicing under hypoxia plays important roles in the spliceosomal proteins-associated autosomal dominant retinitis pigmentosa.Together,we found that PRPFs(pre-mRNA processing factors)play an important role in mitophagy,and a group of PRPFs mutated in retinitis pigmentosa is required for mitophagy,including BRR2,PRPF6,PRPF8 and PRPF31.In conclusion,PRPF8 is a novel regulator by regulating ULK1 mRNA splicing and expression during hypoxia-induced mitophagy.Our results suggest that defects of adRP-related PRPFs-mediated mitophagy may contribute to retinitis pigmentosa pathogenesis.In the screening based on the small molecular compound library,we identified several compounds as candidates.CCR4 antagonist(C-C motif chemokine receptor 4 antagonist)is an interesting compound.Our results shown that CCR4 antagonist not only obviously inhibited CCCP-induced mitophagy and hypoxia-induced mitophagy,but also significantly blocked autophagic flux under basal conditions.Consistent with this,we further observed that CCR4 antagonist induce LC3 puncta that colocalized with lysosome using fluorescence microscope.Meantime,CCR4 antagonist also induces cell death that depends on autophagy,because autophagy inhibitor Baf A1 was able to restore the cell viability.However,more efforts are needed to further explore the mechanisms of CCR4 antagonist on autophagy.In this study,we constructed mitophagy detection technology and high-content screening system based on mtKeima,identified several regulatory molecules of mitophagy and revealed its mechanism.Our data will offer a potential therapeutic target and strategy to treat the diseases because of dysregulation of mitophagy. |
