Cleavage Of MicroRNA By Zebrafish Drosha And Drosophila Dicer And The Effects On MicroRNA Expression At The Genome-Wide Level

MicroRNAs(miRNAs)are a large family of approximately 22 nucleotides long RNAs that regulate gene expression.Their biogenesis involves transcription and subsequent processing steps to produce single-stranded mature miRNAs.Among them,Drosha and Dicer are two key enzymes that cleave primary miRNA(pri-miRNA)and precursor miRNA(pre-miRNA)transcripts,respectively.Although human Drosha,Dicer and Drosophila Dicer have been reported,the mechanism of action and substrate specificity of Drosha/Dicer remains unclear.Therefore,at the genome level,we conducted a corresponding study on zebrafish Drosha and Drosophila Dicer.In this study,we cloned and constructed expression plasmids encoding zebrafish Drosha,and its accessory protein DGCR8.In vitro cleavage of 105 pri-miRNAs by active zebrafish Drosha/DGCR8 revealed that different miRNAs have different cleavage efficiency.The analysis of the pri-miRNA acting on the zebrafish Drosha/DGCR8 is as follows:With the increasing of the number of base pairings(bp)in the dsRNA(double-stranded RNA)region and nucleotide(nt)in the terminal loop,the cleavage efficiency of zebrafish Drosha/DGCR8 to pri-miRNA also increased;The stronger the stability of the proximal domain,the higher the cleavage efficiency of Drosha/DGCR8 cleavage,so the miRNA expression will be enhanced.miRNAs are generally numbered according to the chronological order of discovery,that the later discovered miRNAs were more likely to be inefficiently processed and expressed suggests that miRNA discovery has approached its limit.Differential analysis of zebrafish-specific miRNAs with Drosophila or human-associated miRNAs revealed that evolutionarily conserved miRNA can be better cleavaged by zebrafish Drosha/DGCR8,and the expression of conserved miRNAs is higher.We used the Drosophila Dicer-1/R3D1-L to perform in vitro cleavage experiments on 120 pre-miRNAs,and different miRNA have different cleavaging efficiency.Analysing the cleavaging efficiency of Drosophila Dicer-1/R3D1-L and structure of pre-miRNA,it was found that the canonical structure consisting of the paired 5' end and the 2-nt 3' overhang was not necessary,and the large terminal loop enhanced pre-miRNA cleavage.The stability of the pre-miRNA,dsRNA region and terminal loop region was not significantly correlated with the cleavage efficiency of Drosophila Dicer-1/R3D1-L,but the stronger the stability of pre-miRNA and dsRNA regions,the higher the expression level of miRNA.It was reported that miRNA found later were more likely to be inefficiently cleavaged by Drosophila Dicer-1/R3D1-L,the expression level of miRNA is also lower.Through analyzing Drosophila miRNA and zebrafish or human miRNAs,we found no significant difference in the cleavage efficiency of Drosophila conservative and non-conservative miRNA by Drosophila Dicer-1/R3D1-L.However,conserved miRNAs have higher expression levels than non-conservative miRNAs.Taken together,our research lays the foundation for future research on the regulatory and conserved mechanisms of Drosha and Dicer.