The Structural And Functional Investigations Of The KH Domains Of Human E3-ligase MEX-3C

The specific RNA-protein interaction plays an essential role in gene regulation,including transcriptional control, RNA processing and transport, mRNA degradation and translational control. RNA-binding proteins interact with the RNA targets via their RNA-binding domains (RBD), such as the double-stranded RNA-binding domain(dsRBD), RNA recognition motif (RRM), zinc finger domain and hnRNP K-homology domain (KH).The KH domain was originally identified in human heterogeneous nuclear RNP K(hnPNP K) and is one of the most abundant RNA binding domains in all three life domains. Multiple copies of the KH domain are often observed in the KH domain-containing protein, which can enhance protein-nucleic acid binding affinity and speci-ficity. The KH domain consists of approximately 70 amino acid residues, that form a structure in which three a -helices pack against a β-sheet composed of three antipar-allel β-strands. Based on the topological arrangement, KH domains can be classified into two versions, named type I and type II KH fold. The type I fold typically ex-ists in eukaryotic proteins, whereas the type II typically exists in prokaryotic proteins.Both types form a hydrophobic cleft that accommodates up to four single-stranded nu-cleotides. KH domain-containing proteins perform a wide variety of cellular functions,and the interaction between the KH domain and their RNA substrates is essential for the establishment of post-transcriptional regulatory networks.The Caenorhabditis elegans MEX-3 is one member of the KH domain-containing protein family. MEX-3 contains two KH domains and plays an essential role in early embryonic development and the maintenance of germline totipotency. A family of four mammalian homologs to the C. elegans MEX-3 (MEX-3A-D) was subsequently identified in human and mouse. They all contain two KH domains and one ubiquitin E3-ligase RING finger domain in the carboxy-terminus. These four mammalian homologs are thought to play similar roles as their C. elegans ancestor in post-transcriptional regulation. Of these four homologs, MEX-3C has been reported to be associated with several diseases, including postnatal growth, hypertension, energy metabolism, immune responses, and cancer. Although the° human MEX-3C has been demonstrated to be involved in translational regulation of many mRNAs, HLA-A2, to the best of our knowledge, is the only one that has been shown to bind directly to hMEX-3C KH domains. MEX-3C inhibits the translation of the HLA-A2 mRNA by binding to its 3’ UTR. Moreover, in the presence of RING domain, MEX-3C can also induce the ubiquitin-dependent degradation of this mRNA. However, the molecular mechanisms of mRNA degradation and translational repression induced by MEX-3C, as well as the consensus RNA sequence recognized by the MEX-3C KH domains remain to be elucidated.In this study, we determined the high-resolution crystal structures of hMEX-3C KH1-GUUUAG and KH2-CAGAGU complexes. Based on the structures, we generated a series of alanine mutants for ITC analysis °and then revealed the crucial residues involved in RNA-protein interaction. Furthermore, we also determined the consensus RNA motifs for MEX-3C KH domains. Based on these results, we mapped the real RNA targets for the hMEX-3C KH domains at the 3’ UTR of HLA-A2 mRNA, which was further confirmed by fluorescence polarization (FP).