Alternative Polyadenylation And Post-transcriptional Regulation Of Bovine AAMDC Gene

Post-transcriptional regulation,including the processing,export,localization,turnover and translation of mRNA,is an integral part of gene expression.In recent years,it has become increasingly evident that APA which yields transcript isoforms with variable3'UTRs is emerging as an important regulator contributed to gene expression.APA has a significant impact on localization,stability and translation efficiency of mRNA.The bovine AAMDC(Adipogenesis associated Mth938 domain containing)gene is located on chromosome 29,contains 4 exons and encodes a 122-aa protein.Conserved domain analysis reveals that AAMDC contains a predicted Mth938 domain,which is a hypothetical protein domain encoded by the Methanobacterium thermoautotrophicum(Mth)genome.The function of the domain has not been determined.The post-transcriptional events regulating AAMDC expression,however,remain to be elucidated.In this study,3'RACE was performed to define the bovine AAMDC isoforms.First,Luciferase assay was performed to explore the potential effect of the different AAMDC3'UTRs due to APA in regulating protein expression.Moreover,we investigated the effect of 3'UTRs on mRNA stability and translational efficiency by constructing and transfecting the expression vectors.We also explored the interaction of AAMDC gene with microRNAs(miRNAs)by qRT-PCR and Western blot.Finally,the expression profile of AAMDC short and long isoforms during the course of bovine preadipocytes differentiation and its effect on differentiation of bovine preadipocytes were analyzed.The main results are as follows:(1)Bovine AAMDC expresses three isoforms as a result of APA and AS(Alternative splicing,AS).Accession numbers of three isoforms in NCBI are MK576046,MK576047 and MK576048,respectively.(2)Luciferase assay revealed that AAMDC short and long 3'UTR displayed different regulatory role,and the short 3'UTR enhanced the expression of protein.(3)By transfecting expression vectors into HEK293 cells,qRT-PCR and Western blot revealed that mRNA stability and translation efficiency of short isoform was higherthan that of the other isoforms.(4)miR-2428 and miR-664 a directly bind to the 3'UTR of long isoform and cause a decrease in its expression,indicating this APA-mediated shortening of the AAMDC 3'UTR renders the short isoform insusceptible to miRNA-mediated suppression due to loss of the binding sites for miR-2428 and miR-664 a.(5)The expression profile of AAMDC short and long isoforms during the course of bovine preadipocytes differentiation were analyzed by qRT-PCR.Our results showed that the expression levels of AAMDC short and long isoforms,long isoforms were significantly upregulated after adipogenic induction.By transfecting short and long isoforms into bovine preadipocytes,oil Red O staining and qRT-PCR suggested that short AAMDC isoform promoted the differentiation of bovine preadipocytes.Collectively,our study elucidated a novel post-transcriptional regulatory mechanism of AAMDC,including APA,miR-2428 and miR-664 a.We also demonstrated that the short AAMDC isoform promoted the differentiation of bovine preadipocytes,which present a solid foundation for further study into the physiological function of AAMDC and mechanism of adipogenesis.