Screening Proteins Of Interest Interact With Dsmurf Via Yeast Two Hybrid System

Friut fly is one of the most outstanding model species for scientific research. Many molecular biological mechanisms from flies to human is highly conserved during evolution. Unlike mammals, drosophila is more simple and convenient. We would like to choose this model to clarify protein interactions on the molecular level, and hopes to promote molecular regulation mechanisms and related diseases of other higher species or humans.Smurf is a kind of ubiquitin E3 ligases, which belongs to Nedd4 family. Smurf negatively regulate TGF-β(BMP in fly) signaling, and also involved in other signaling pathway. It plays a very important role in development and stem cell mantainance. With respect of the significance, we choose this gene to construct a bait for screening via yeast two hybrid.The screen result shows that there are 40 different proteins have the interational relationship with smurf, we are interested in Rpl 18A and Rpl 24. Ribosomal proteins have crucial roles in life process. Generally speaking, they assemble to an integrated ribosome to decode mRNAs into proteins. In addition, many RPs also fill various roles that are independent of protein biosynthesis, called extraribosomal functions, which including DNA replication, transcription, DNA repair, RNA splicing and modification, cell growth, proliferation, apoptosis and development regulation, cellular transformation and others. RPs mutation can cause cancer and diamond blackfan anemia (DBA).Through the preliminary yeast two hybrid experiments, we find that smurf could interact with Rpl 18A/24, and notice that the mutant phenotypes between smurf15c and RPs are similar. Immunofluorescence experiment shows that smurf colocalized with Rpl 18A/24, and further study indicates that smurf can physically interact with Rpl 18A/24. It has been reported that ubiquitination could promote RPs to assemble ribosomes, and enhance the translation efficiency of ribosome. In conclusion, we have an hypothesis: Rpl 18A/24 ubiquitinated by smurf can promote them to ribosomes and enhance the mRNA decoding efficiency. But how does smurf ubiquitinate RPs on molecular level? Whether Rpl 18A/24 mutant will show the similar phenotype in germline stem cell? To solve these questions we need further our study.