Identification Of Novel Isoforms Of The MYNN Gene And Their Function Preliminary Study In Pig

Myoneurin(MYNN)belongs to the BTB/POZ and zinc finger protein family whose members play roles in the activation and repression of transcription,cytoskeleton organization,and chromatin remodeling.Alternative splicing is a major source of protein diversity in organisms,and greatly influences the structure and function of proteins.MYNN gene alternative splicing(AS)have been found in human and mouse,and MYNN can regulate gene expression in the human muscle.However,the studies on alternative splicing and their fu.nctions of the MYNN gene have not been reported in pig.Therefore,this study focused on Large White and Mashen pigs,using RNA sequencing,PCR and cloning to identify the novel transcripts of the MYNN Gene in pig muscle,and the expression profiles of MYNN-1 and MYNN-2 in 11 different tissues including heart,liver,spleen,lung,kidney,cerebellum,small intestine,stomach,pancrease,longissimus dorsi,fat of Large White and Mashen pigs at 90 days and the developmental expression patterns in different tissues at three stages(1,90,180 days)were studied by quantiyative Real-time PCR(qPCR).The results shown that:(1)We identified two novel transcripts of the MYNN Gene in pig muscle,named MYNN-1 and MYNN-2 respectively.The coding sequence(CDS)region of the two isoforms was cloned in the muscle of pigs.MYNN-1 was 84 bp longer than MYNN-2,and MYNN-2 lacked the sixth exon.The splice sites complied with the GT-AG rules at the 5’ splice donor and 3’ splice acceptor sites.Pig MYNN-1 CDS is composed of 1830 nt encoding 609 amino acids and include 8 exons,whereas MYNN-2 CDS is composed of 1746 nt encoding 581 amino acids and include 7 exons.There are four ZF-C2H2(zinc finger,C2H2-type)in the 84-nucleotide sequence,which has a deletion mutation in the MYNN-2 variant.(2)MYNN-1 and MYNN-2 were ubiquitously expressed in pig tissues,which include heart,liver,spleen,lung,kidney,cerebellum,small intestine,stomach,pancrease,longissimus dorsi,fat,and there were significant differences in different tissues.In the same breed,the expression patterns of MYNN-1 and MYNN-2 were the same.MYNN-1 and MYNN-2 all had the lowest expression in fat.In Large White,the expression of MYNN-1 and MYNN-2 in cerebellum was the highest,followed by liver,stomach,spleen and kidney.where in Mashen pigs,the expression of MYNN-1 and MYNN-2 in stomach was the highest,followed by spleen,cerebellum,small intestine,liver and kidney;In different breeds,the expression of MYNN-1 and MYNN-2 in liver,spleen,kidney and fat of Large White was significantly higher than that in Mashen pigs,where in the lung,small intestine,stomach,pancreatic tissues had the opposite results;The expression of two isoforms of MYNN was higher in the Cerebellum,liver,pancreas,kidney,stomach,spleen and lung,and there were significant differences in different tissues.(3)In order to further study the function of MYNN in pig muscle,we examined the developmental expression of MYNN-1 and M-YNN-2 in three muscle tissues,the result shown that:The expression of two isoforms of MYNN were significant difference in different developmental stages of pig tissues,the regular of developmental expression of MYNN-1 and MYNN-2 in longissimus dorsi,biceps femoris and psoas major are different,while on the whole has the same trend.The expression patterns of MYNN-1 and MYNN-2 were similar in the same pigs.In Large White,the expression of MYNN-1 and MYNN-2 were higher in 1-90 days of age,and relative low in 90-180 days of age;The regular of Mashen pigs were opposite with Large White.(4)The expression of two isoforms of MYNN was higher in the Cerebellum,liver,pancreas,kidney,stomach,spleen and lung,and there were significant differences in different tissues by analysing the expression profile of MYNN-1 and MYNN-2.So we further study developmental expression patterns of two novel isoforms in Cerebellum,liver,pancreas,kidney,stomach,spleen and lung tissues of pigs:The developmental expression patterns of two variants of MYNN in the cerebellum,liver and stomach had the same regular,The expression of MYNN in three developmental stages(1,90,180)of Large White were gradually increased,while in Mashen pigs were opposite.There are certain rules in the three stages in other tissues,which is related to the specific function of MYNN gene in specific tissues.(5)The relative expression of MYNN-1 was significant higher than MYNN-2 in different tissues of Large White and Mashen pigs at 90 day old stage.Therefore,MYNN-1 is the main isoform of pig MYNN.The study of the developmental expression patterns of tissues in pigs,we found that MYNN-1 is the main isoform in muscle,cerebellum,liver,spleen,pancreas,stomach,kidney,lung tissues of Mashen pigs and longissimus muscle,psoas muscle,liver,spleen,cerebellum,pancreas,stomach tissues Large white pigs,while in biceps femoris,lung and kidney tissues of Large white pigs,MYNN-2 is the main isoform of pig MYNN.To sum up,there are two novel alternative splicings of MYNN in pig muscle tissue,and MYNN-1 was the main isoform.Those two isoforms of MYNN may play an important role in the muscle,cerebellum,liver,spleen,pancreas,stomach,kidney,and lung tissues of pigs,and their functions are related to the tissues,the developmental stages and the genetic background of the pig breeds.