Structural And Functional Insights Into Fission Yeast Protein Mei2 And Human Protein ERH

Non-coding RNA is a type of RNA molecule that is widely present in organisms and cannot be translated into protein but has important biological functions.Non-coding RNA participates in intracellular processes such as translation,RNA splicing,DNA replication,and cell cycle gene regulation.Non-coding RNA is associated with many diseases,such as cancer and Alzheimer’s disease.However,there are still many unknowns in the study of non-coding RNA.The study of the structure and function of non-coding RNA and its related proteins has important biological significance.This paper contains two parts,one is that the structural and functional insights reveal the specific recognition of meiRNA by the Mei2 protein in fission yeast;the other is that ERH facilitates microRNA maturation through the interaction with the N-terminus of DGCR8.In the fission yeast Schizosaccharomyces pombe,Mei2,an RNA-binding protein essential for entry into meiosis,regulates meiosis initiation.Mei2 binds a specific noncoding RNA species,meiRNA,and accumulates at sme2 gene locus,which encodes meiRNA.Previous research has shown that the Mei2 C-terminal RRM(RRM3)physically interacts with meiRNA 5’ region in vitro and stimulates meiosis in vivo.However,the underlying mechanism still remains elusive.We first employed an in vitro CLIP-seq assay and demonstrated a preference for U-rich motifs of meiRNA by Mei2 RRM3.We then solved the crystal structures of Mei2 RRM3 in the apo form and complex with an 8mer RNA fragment,derived from meiRNA,as detected by in vitro CLIP-seq.These results provide structural insights into Mei2 RRM3-meiRNA complex and reveal that Mei2 RRM3 binds specifically the UUC(U)sequence.Furthermore,a structure-based Mei2 mutation,Mei2F644A,which disrupts the Mei2-meiRNA interaction,causes defective karyogamy,suggesting an essential role of the interaction between Mei2 and meiRNA in regulating meiosis.The Microprocessor complex processes the primary transcript of microRNA(primiRNA)to initiate miRNA maturation.Microprocessor is known to consist of RNase III Drosha and dsRNA-binding protein DGCR8.Here we identify Enhancer of Rudimentary Homolog(ERH)as a new component of the Microprocessor.Through a crystal structure and biochemical experiments,we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8,in a 2:2 stoichiometry.Knock-down of ERH or deletion of the DGCR8 N-terminus results in a decrease of processing of primary miRNAs with suboptimal hairpin structures that reside in polycistronic miRNA clusters.ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144.Thus,the ERH dimer may mediate "cluster assistance" in which the Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster.Our study reveals a role of ERH in the miRNA biogenesis pathway.In summary,we determined the structure of the Mei2 RRM3 using a natural 8mer meiRNA fragment identified by in vitro CLIP-seq.We found that the specific recognition of meiRNA by Mei2 RRM3 is indispensable for the meiosis process.We also identified the binding preference of Mei2 RRM3 for the UUC(U)motif in vitro.In addition,we determined the structure of the ERH in complex with DGCR896-126 peptide and revealed the molecular basis in the maturation process of non-coding RNA miRNA.