Functional Analysis Of The Insertion Of L1 From The Double-stranded RNA Binding Domain And Differential Inhibition Of Target Gene Expression By Human MiR-1

MicroRNAs(miRNAs)are a class of non-coding RNAs that are widely expressed in organisms and are approximately 22 nucleotides in length.In the canonical pathway,miRNA is generated by consecutive actions of two RNase III enzymes(Drosha and Dicer).Drosha,together with its partner protein DGCR8,is in a complex known as the Microprocessor,which cleaves precursor transcripts called primary miRNA(primiRNA)to produce hairpin precursor miRNA(pre-miRNA).Drosha contains a Cterminal double-stranded RNA-binding domain(dsRBD).NMR analysis revealed that the dsRBD from Drosha(Drosha-dsRBD)has a α1-L1-β1-L2-β2-L3-β3-L4-α2structural fold,which is similar to other dsRBDs structures.The difference is that the L1 region from Drosha-dsRBD has a short insertion about 6 amino acids,compared to other dsRBDs.The biological function of the insertion of the L1 region on DroshadsRBD is unclear.In addition,dsRBDs usually have certain variability particularly at the α1 and L2 regions.Why is there no similar insertion in all other dsRBDs,and will the L1 insertion disrupt their functions?In animals,miRNAs play an important role in different development and pathological processes by inhibiting their target genes expression.Therefore,identifying miRNA targets is the key to understanding the miRNA function.At present,miRNA target genes are often obtained through various prediction platforms,but most of the predicted target genes have not been confirmed.Therefore,most miRNA target genes are unknown,and the degree to which a miRNA differentially inhibiting the expression of its targets is underappreciated.we conducted the following study on miRNA synthesis and function to investigate this problem.On the one hand,we aim to show that the insertion of the L1 region in the Drosha-dsRBD is essential for its function during the miRNA synthesis process,hoping to provide a theoretical basis for the interpretation of Drosha’s processing of RNA.At the same time,the variability of the L1 region in dsRBD can be revealed,which further proves the diversity of dsRBD structure.On the other hand,we select human miR-1 as a representative specifically expressed in the human heart.And explorations are made regarding how miRNAs can inhibit their targets to varying degrees and affect the expression of endogenous genes.The results suggest that the differential expression of endogenous genes may partly be attributed to the regulated miRNA.The major results are summarized as follows:1 The Weakened dsRBD Ability to Bind RNA and Drosha Efficiency to Cleave PrimiRNA Caused by the Deletion of Unique Extension in the L1 RegionWe constructed a series of point mutations and small deletions of Drosha according to the 6 amino acids insertion in the L1 region from Drosha-dsRBD.The human pri-miR125 b and pri-miR-31 are selected as representatives to investigate the pri-miRNA processing activity by all Drosha mutations.It is found that a number of point mutations and,especially,deletions in L1 significantly reduced Drosha processing of pri-miRNAs.The processing activity of the mutants are only 10% ~ 50% of the wild-type protein.In addition,it is shown that the dsRBD of Drosha exhibited a weak yet noticeable affinity for RNA,and that the deletion mutant in unique extension(Δ6)of dsRBD showcased a lower RNA binding ability than the WT in vitro.By using si RNA to reduce the expression of endogenous Drosha protein in cells,it is demonstrated by reporter assays and q PCR results that the expression of Drosha(Δ6)mutants in cells did not significantly improve the expression of miRNA.Therefore,the study results indicated that the insertion of L1 region in the Drosha-dsRBD is important for its function.2 The Influence of Insertion in the L1 Region of Other dsRBDs on Their FunctionsThe 6 amino acid sequences from the L1 region of Drosha-dsRBD were inserted into the dsRBD structural domains of the Xlrbpa,Rnt1,Staufen,human Dicer,and DGCR8 proteins.And the ability of dsRBDs to bind RNA in vitro was tested through gel mobility shift analyses.The results showed that such ability was significantly reduced.In addition,L1 insertion in dsRBDs also adversely affected the activities of full length DGCR8 and Dicer proteins.Reporter assays and q PCR also showed that the extension of the L1 region in Dicer-dsRBD can inhibit the function of Dicer in human cells.Our results add to the growing appreciation of the diversities in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to variations in the L1 region.3 Confirmation of Mi R-1 Predicted Target GenesMammalian miRNAs reduce the protein translation level and/or mRNA expression level of the target genes primarily through Watson-Crick pairing between the miRNA and the miRNA-response element(MRE)in the 3’ UTRs of target mRNAs.We constructed miRNA target gene reporter libraries based on prediction programs Target Scan,miRanda,and Pic Tar,and performed large-scale reporter assays to directly evaluate whether and how strongly a predicted target gene is repressed by the miR-1.The results showed that 162 of the 196 predicted target genes were verified as true miR-1 target genes by the reporter assays,and different reporters were inhibited to different extent by miR-1.We deleted mRNA sequences complementary to the miRNA seed region in select confirmed targets for miR-1,and then compared the responses of wildtype(WT)and mutant(MUT)reporters to miR-1.The degree of repression of the MUT reporters by miR-1 are significantly lower than that of the WT reporters,indicating the dependence of miRNA inhibition on MREs.4 Effects of miRNA Differential Repression of Target Genes on the Endogenous Genes ExpressionmiR-1 is specific and highly expressed in heart.By comparing the relationship between the results of reporter assays and the mRNAs expression in human heart,it is found that the repression of reporters by miR-1 has a certain correlation with the expression of corresponding mRNA in the ventricle(r=0.123,p < 0.05),indicating that the endogenous gene expression is partly due to the differential target repression of miRNAs In addition,how the expression of endogenous target genes was affected by miR-1 overexpression in human cells is investigated.We transiently transfected miR-1 mimic into 293 T cells,determined the global mRNA expression by RNA-seq,and then explored how the confirmed targets changed their mRNA expression.It was discovered that the results of reporter assays and the degrees of mRNA expression changes had a positive and significant correlation,consistent with the idea that differential miRNA repression influences endogenous gene expression.5 Effects of the Secondary Structure of Target Gene mRNAs and the Interaction Between miRNA and mRNA on the Degree of miRNA Inhibition of its TargetsAccording to the above experimental results,it is found that the inhibition of miR-1 on its target genes varies in extent.In this study,we gathered a large amount of data that may affect the degree of miRNA inhibition of its targets,by means of search,collection and calculation.By using statistical methods,we identified parameters that could predict the miRNA repression degrees.The results showed that: 1)the more extensive the MRE of target and miRNA seed region are paired,the stronger the inhibition of target will be.2)the conserved sites were slightly better confirmed as miRNA targets,with its inhibitory effect significantly stronger than poorly conserved sites(t-test,p < 0.05).3).In addition,the AU% and minimum free energy(MFE)of the predicted secondary structure of cloned target 3’ UTR is prominently correlated with the degree of reporter inhibition(r= ~ 0.2,p < 0.05),suggesting that the more flexible the mRNA is,the more inhibition of target will be by miRNA.4)the binding energy between miRNA and MREs is in positive correlation with the degree of reporter inhibition(r=0.237,p < 0.05),indicating that tighter binding contributed to stronger inhibition of target by miRNA.