The Structural And Functional Analysis Of The N-terminal Tandem RRM Domains Of Human RBM45

RBM45 is an RNA-binding protein involved in neural development,whose aggregation is associated with neurodegenerative diseases amyotrophic lateral sclerosis(ALS)and frontotemporal lobar dementia(FTLD).RBM45 contains three classical RRM domains,all three of which have the ability to bind RNA,RBM45 can bind RNA in a sequence-specific manner.However,the binding of RBM45 with specific sequence RNAs and the mechanism of RBM45 aggregation in the cytoplasm of ALS and FTLD neurons are still unknown.In this study,the three RRM domains of RBM45 were first divided into the N-terminal tandem RRM1 and RRM2 and the C-terminal RRM3.we resolved the crystal structure of the N-terminal tandem RRM domains of human RBM45 in complex with single-stranded DNA(ss DNA)by using X-ray crystallography.The structure of the complex showed that the RRM1 bound GACGG-ss DNA and RRM2 bound GGACG-ss DNA,both of which contained the GAC motif.The affinity of the two RRM domains to the specific sequence RNA was determined by Bio-layer interferometry assay.Two aromatic residues and an arginine residue in each RRM were critical for RNA-binding,and the inter-domain linker was also involved in RNA-binding.The N-terminal tandem of RBM45 with two RRM domains formed a pair of antiparallel RNA binding sites,and by multi-angel light scattering it was determined that the N-terminal tandem RRM domains bound separate GAC motifs in one RNA strand or GAC motifs in different RNA strands,respectively.The results of this study will contribute to identify the physiological targets of RBM45 and provide a basis for understanding the physiologic and pathologic functions of RBM45 in neural development.