Dynamic Changes Of 3’UTR In Porcine Testicular Sertoli Cells Induced By Acute Heat Stress

The location,processing,transportation and translation of mRNAs are important to regulate gene expression.The 3’untranslated regions(3’UTR)of mRNAs affect biological processes of mRNA stability,subcellular localization and translation efficiency.If the length of 3’UTR changes,gene expression could be abnormal due to differences of cis regulatory elements contained,therefore cause disease or affect animal production performance.Alternative polyadenylation(APA)can form 3’UTR isomers with different lengths at the 3 ’end of mRNA,thus affect the gene expression at post-transcriptional level.Acute heat stress can rapidly affect gene expression,however,it is still unclear for detail molecular regulatory mechanism.In this study,porcine immature sertoli cells(iSCs)were taken as the research object.Based on transcriptome sequencing data,bioinformatics analysis identified the dynamic changes of 3’UTRome during and after acute heat stress,and verified the regulation of gene expression by the 3’UTR isomer of key gene,providing references for 3’UTR to regulate the effects of acute heat stress on animal male reproduction.The main results as follows:(1)3’UTRome changes induced by acute heat stress of porcine iSCsThe transcriptome sequencing data of porcine iSCs before and after acute heat stress(Control group: before heat stress;HS0.5: 43 ℃ heat stress for 0.5h;HS0.5-R36: 36 h after heat stress at 43 ℃ for 0.5h)were analyzed,using bioinformatics software Da Pars and APArap to identify the dynamic changes of APA events.Da Pars identified 639(HS0.5 vs.Control),464(HS0.5-R36 vs.HS0.5)and 290(HS0.5-R36 vs.Control)mRNAs with3’UTR significant differences,while APArap identified 713,518 and 321 mRNAs with3’UTR significant differences.The mRNAs with significant different 3’UTRs obtained by Da Pars are mainly enriched in GO terms of microtubule,mitochondrial matrix,intercellular junction,protein domain specific binding and steroid binding etc.;KEGG pathways of pyruvate metabolism,glycolysis/gluconeogenesis,apoptosis,P53 and HIF-1 signal pathways,etc..The mRNAs with significant different 3’UTRs obtained by APAtrap are mainly enriched in GO terms of positive regulation of intrinsic apoptosis,antioxidant activity,ATP metabolic process,translation,RNA binding etc.and in KEGG pathways of PI3K-Akt,P53,apoptosis,AMPK,Hippo,glycolysis/gluconeogenesis etc.Further analysis screened out 3’UTR length of Acss2,Inpp1 and Nr1h4 mRNAs to be with longer variation,whose 3’UTR isomers with long and short sequence contained different cis regulatory elements(CPE and PAS,micro RNA-binding sites and RNA-binding protein sites);the 3’UTR abundance of Hsp70.2,Inhbb and Dhrs7 mRNAs have significant differences during acute heat stress response of porcine iSCs.These results provide references to further uncover the molecular regulatory mechanism of 3’UTRs during acute heat stress response of porcine iSCs.(2)Screening stable RT-qPCR internal reference(s)in porcine iSCs under acute heat stresTo RT-qPCR validate mRNAs with 3’UTR significant differences in length or abundance as identified in(1),the stability of six candidate internal reference genes(Hprt1,Top2 b,Actb,Rpl32,Gapdh and 18S)in porcine iSCs under acute heat stress response(Control,HS0.5 and HS0.5-R36 groups)was systematically evaluated.The analysis results of Ge Norm,Norm Finder,Best Keeper,Comparison ΔCt and Ref Finder showed that Rpl32 and Actb were the most stable internal references,while Hprt1 and 18 S were the most unstable internal references.The results provide suitable internal reference gene(s)to normalize the expression of transcripts in porcine iSCs under acute heat stress.(3)Identification,expression and function of 3’UTR isomers of key genes in porcine iSCs under acute heat stress3’RACE confirmed that 3’UTRs of Acss2 and Nr1h4 mRNAs were with different isoformers,but Inpp1 was without 3’UTR isoformers in porcine iSCs during acute heat stress.Acss2 has two 3’UTR variants,and successfully sequenced two 3’UTR sequences(94 and 384nt)(3’UTR-S and 3’UTR-L).Four 3’UTR variants existed in Nr1h4,and three3’UTR sequences were sequenced successfully(53,264 and 371nt)(3’UTR-S,3’UTR-Ler and 3’UTR-L-est).Semi-quantitative RT-PCR and quantitative RT-qPCR(normalized using selected internal reference genes in(2))showed that two 3’UTR transcripts of Acss2 and three 3’UTR transcripts of Nr1h4 were expressed during acute heat stress response of porcine iSCs;during the whole response process,the transcripts of Acss2 3’UTR-L were significantly higher than 3’UTR-S,and the transcripts of 3’UTR-L-er and 3’UTR-L-est of Nr1h4 were also significantly higher than 3’UTR-S;the abundance of 3’UTR-L and 3’UTRS transcripts of Acss2,the 3’UTR-S and 3’UTR-L-est transcripts of Nr1h4 in HS0.5-R36 group were significantly higher than those in Control and HS0.5 groups,and the abundance of the 3’UTR-L-er transcript of Nr1h4 in the HS0.5-R36 group were significantly higher than that in Control group.During the response to acute heat stress of porcine iSCs,mRNA and protein expression of Acss2 was significantly changed,with the abundance in HS0.5-R36 group to be significantly higher than that in Control group.Dual-Luciferase Reporter assay showed that 3’UTR-L of Acss2 inhibited expression of the luciferase reporter gene as compared to3’UTR-S;miR-140-3p could bind to 3’UTR-L of Acss2 to inhibit expression of the luciferase reporter gene;The binding of Igf2bp2 to Acss2 3’UTR-L increases the protein level of ACSS2,whether the effect of Igf2bp2 to Acss2 3’UTR-L on the luciferase reporter gene still needs further confirmation.To sum up,this study systematically analyzed the 3’UTRome of porcine iSCs under acute heat stress,screened and experimentally validated the existence of 3’UTR isomers with different lengths of important genes,screened stable RT-qPCR internal reference genes,detected the dynamic expression patterns of different 3’UTR isomers of important genes,analyzed that report gene expression was inhibited by miR-140-3p binding onto 3’UTR-L of Accs2,and the binding of Accs2 3’UTR-L with Igf2bp2 promotes the protein expression of Acss2.These results revealed the molecular mechanism of 3’UTR regulating the acute heat stress response of porcine iSCs at the post-transcriptional level.