Trans-spliced RPGR-EEF1A1 Promotes Autophagy Via Enhancing RPGR Interaction With RAB37

An accurate splicing of pre-m RNA is a key step of post-transcriptional regulation.In comparison with cis-splicing,there are a few studies concerning trans-splicing,particularly splicing patterns,mechanisms and evolution.Novel RNAs produced by trans-splicing not only increase the complexity of proteome,but also provide new regulation mechanisms of gene activities.As a catabolic process,autophagy can degrade composition of the cytoplasm and organelles through the autophagosome and lysosome,and autophagy injuries cause many human diseases.However,autophagy regulation through trans-splicing has not been reported so far.The role of trans-splicing in autophagy is of great significance to understand functions of autophagy in both physiological pathological processes.In this study,using RNA-seq databases and experimental verification,trans-spliced Rpgr-Eef1a1 molecule was identified.The molecule contains a common repetitive sequence in splicing site,indicating that it was spliced via homology and conforms to the transcriptional slippage model.In vitro simulation experiments,four endogenous transcripts and another two simulated transcripts were obtained.The transcripts,Rpgr-Eef1a1 a and Rpgr-Eef1a1 b,contain vector sequence.All of the transcripts meet "GU-AG" splicing role,RIP analysis showed that spliceosome was involved in trans-splicing process.Protein Co-IP from samples of mouse ovary and retina showed that RPGR can interact with RAB37 and also showed GEF activity.In GTP enzyme activity detection,RPGR showed its activities only when RAB37-GTP and-GDP coexist.Over-expressed RPGR in wild-type He La cells can promote autophagy,but not when RAB37 was knocked down,suggesting that RAB37 is an effector of autophagy,and RPGR promoted the autophagic process.Protein Co-IP showed that trans-spliced RPGR-EEF1A1 can enhance RPGR interaction with RAB37.RPGR-EEF1A1 contains a complete NB domain and a part of RLD domain(RLD 1C),that RPGR-EEF1A1 has the same GEF function as RPGR protein.The GTP enzyme activity detection experiments also showed the same results.Immunofluorescence and western analysis showed that overexpression of RPGR-EEF1A1 in wild-type He La and mi R-Rab37 cells can promote autophagy via enhancing RPGR interaction with RAB37.These data suggested that RPGR-EEF1A1 can enhance RPGR interaction with RAB37,thus promote autophagy.