Identify RNA Helicase Associated RNAs By GoldCLIP-Seq In The C.elegans Germline

Protein-RNA interactions widely exist in cells,and are crucial for most biological processes in all organisms.Therefore,it is very important for studying cell functions and molecular mechanisms at the individual level to obtain tissue specific protein-RNA interaction information under physiological conditions,which is still one of the technical difficulties in this field.In this study,we modified GoldCLIP(Gel-omitted Ligationdependent CLIP),a method previously developed in cultured cells,to make this technology applicable in C.elegans,which expands the toolkit for studying tissue specific protein-RNA interactions at the animal individual level.GoldCLIP is a technology dependent on HaloTag protein tag,which covalently binds to a synthetic ligand.The GoldCLIP technology can minimize non-specific RNA binding and reduce RNA loss,facilitating downstream generation of RNA sequencing library.In addition,GoldCLIP technology does not require the use of radioactive reagents,which improves the safety of experimental operations.We verified the reliability of GoldCLIP in C.elegans by studing two RNA helicases,GLH-1 and HIP-1,two factors discovered in our previous study which regulate the piRNA(PIWIinteracting RNA)gene silencing pathway.We identified various mRNAs which bind to GLH-1,HIP-1 or GLH-1 DEAD domain mutants.These results will help us to further understand how GLH-1 and HIP-1 participate in the piRNA pathway.