Structural Study Of Non-coding RNA And Human Roquin-2's Complex

Non-coding RNAs play an important regulation role in cells.Constitute-decay element(CDE)is a highly conserved stem-loop motif located in the 3' UTR of mRNAs that encode regulators of cell development and inflammation,such as tumor necrosis factor ?,inducible T cell co-stimulatory factor.CTLA4 RNA is a 15 nt non-coding RNA,whose sequence is similar to the reported CDE sequence,but the tertiary structure is unknown.It was reported that Roquin and its homolog Roquin-2 mediate mRNA degradation through binding the CDE of mRNA in cell and play an essential role in RNA homeostasis and autoimmune diseases.Human Roquin-2 is a class of ubiquitin ligase.Its N-terminus mainly contains a ROQ domain and two HEPN domains,of which the ROQ domain is charged for binding RNA.It's unknown whether there is a rule for Roquin's binding CDE and regulating mRNA.In this study,we purified CTLA4 RNA and two constructs(171-325,87-404)of Roquin-2 in vitro,demonstrated that CTLA4 RNA interacts with Roquin-2 by EMSA experiments.The crystal structure of CTLA4 RNA in a complex with Roquin-2(171-325)was determined at 2.15 ? resolution using X-ray crystallography.We found that some key residues(S235,K236,Q244 and S261)of Roquin-2 is vital to bind RNA by interaction analysis.Sequence alignment shows that the RNA is highly conserved in the CDE/Roquin-2 complexes.All the work established the foundation for further study on the regulation mechanism of CDE/Roquin complex.