The Pathogenesis Of Cullin 3 Synonymous Mutations Associated With Familiar Hyperkalemic Hypertension And Targeted Intervention Study

Objective: Pseudohypoaldosteronism type II(PHAII),also called Familiar hyperkalemic hypertension(FHH),is a rare renal tubular disease that is inherited in an autosomal dominant manner.To date,seventeen mutations in CUL3 gene have been identified to be related to PHA II(The Human Gene Mutation Database,up to 2021.2),which all contributed to exon skipping to different degrees.The aim of this study was to investigate the splicing regulatory elements and the interaction between cis-acting elements and trans-acting factors in PHA II underlying the splicing events of c.1221A>G(p.Glu407Glu)identified by us and c.1236G>A(p.Leu412Leu)founded by Boyden etc.Methods: 1.The c.1221A>G and c.1236G>A mutates were evaluated by Human Splicing Finder(HSF)to predict the potential splicing regulatory motifs and associated RNA-binding proteins.2.RNA-pull down assay was performed using biotin-labeled RNA probes and nuclear extract according to the manufacturer’s protocol.And then the eluted RNA-binding proteins were separated by electrophoresis and visualized by silver staining to confirm the proteins.3.Western blotting was carried out to compare the difference between the wild-type and mutant RNA-ESS/ESE and determine the specificity of the trans-acting factor hn RNPx/SRx binding to ESS/ESE.4.The p SPL3-CUL3-E9 WT/Mut minigenes was co-transfected with small interfering RNA or overexpressed plasmid into 293 T cells to verify the involvement of trans-acting factors in the splicing regulation of CUL3 exon 9.5.We conducted site-directed mutagenesis of upstream and downstream of the mutations we studied to determine the key sequences of ESEs or ESSs in m RNA level.6.Rescue experiment was to validate the effect on exon splicing at the protein level underlying the double mutant.Results: 1.The c.1221 A > G mutate was predicted to disrupt all of these ESEs and create a potential ESS for the binding of a negative regulator protein,hn RNP A1(AAGAGG &GAGGTA).In addition,the synonymous mutation c.1236G>A evaluated by HSF was predicted to abolish an ESE recognized by SC35(GGATAAAG)and create a new ESS interacted with hn RNP A1(TAGATA).2.Determine the molecular weight of the target proteins.The results of silver staining showed that the differential protein bands were mainly in the 30-40 k Da region.From the mass spectrometry results,the following proteins were screened for Western blot validation: hn RNP A1,hn RNP A2,hn RNP A3,hn RNP C,SC35,TRA2β,ASF/SF2,and 9G8.3.The results showed that c.1221A>G(Mut1)but not wide-type(WT1)bound very efficiently to hn RNP A1,hn RNP A2,hn RNP A3 and hn RNP C proteins.Meanwhile,no difference was detected in SC35,ASF/AF2,9G8 and Tra2β.c.1236G>A(Mut2)RNA probe did not show significant cross-linking to hn RNP A2,hn RNP C,ASF/SF2,9G8,Tra2β and SC35 protein,whereas it bound weakly with hn RNP A1 and hn RNP A3 proteins.None of the other trans-acting factors were detected in WT2.4.The results of si RNA experiments showed a weak effect on exon inclusion of Mut1 in the presence of single si RNA.The experiment of combined si RNA promoted exon inclusion significantly in comparison with the negative control.On the other hand,downregulation of hn RNP A1 and hn RNP A3 both enhanced the Mut2 splicing of exon 9 from 19% to 35% and 26%,respectively.Overexpression of hn RNP A1 largely boosted exon skipping in p SPL3-CUL3-E9 WT/Mut1 minigene.Otherwise,hn RNP C only enhanced the skipping of exon 9 in the context of Mut1 minigene but did not change the pattern of splicing in wide-type minigene.However,overexpression of hn RNP A3 had no significant effect on p SPL3-CUL3-E9 WT/Mut1 minigene.However,overexpression of hn RNP A1 and hn RNP A3 both largely induced the complete exon skipping in the context of c.1236G>A mutant.5.All variants from positions 12-16 induced the skipping of exon 9 to various extents,indicating that this region seemed to be more in accordance with the generation of ESS than the interruption of ESS.In particular,the variants at positions 17 and 18 in exon 9 resulted in complete exon 9inclusion compared with the wide-type minigene,indicating the generation of a strong ESE.Some nucleotide replacement at positions 27-32 in exon 9 induced exon inclusion in the context of G30 A.6.We verified the effect of A18 G on the splicing regulation of exon skipping induced by A15 G at the protein level.The results showed that binding to hn RNPs family protein was significantly reduced with the new mutation,further demonstrating the role of A18 G in the treatment of exon 9 jumping.Conclusion: In our study,we firstly explored the pathogenesis of exon 9 skipping caused by mutations in the CUL3 gene and verified that the main splicing regulators involved the c.1221A>G and c.1236G>A synonymous mutations are the hn RNPs proteins.In addition,we verified that A18 G can rescue the abnormal splicing caused by the mutation at the m RNA and protein level of splicing regulation,which is meaningful and helpful at the therapeutic level for PHA Ⅱ patients.