The Role Of The Spliceosome Component U2A/SNRPA1 In Spermatogenesis

Maturation of pre-mRNA into mRNA is a very important process during the life of eukaryotes,it is the base of all vital activities.Research in the area of mRN A maturation has passed a long time,and many molecular mechanisms were uncovered.It was mediated by a huge and dynamic clasters of ribonucleoproteins called spliceosome complex.Defects in spliceosome complex can cause a series of genetic diseases However,given the complexity of this process,still little is known of spliceosome complex in development,especially in spermatogenesis.Spermatogenesis is an evolutionarily conserved biological event,which is the foundation for the species to continue.It contains three important processes including spermatogonia proliferation,spermatocytes meiosis and spermatids elongation.Defects in any of the process will cause male reproductive problems.Non-obstructive azoospermia(NOA)is a common disease in male infertility,and it affects about 1%of all adult men worldwide.It is widely known that genetic is one core reason of non-obstructive azoospermia.Our laboratory previously performed genome-wide associated study and found 32 SNPs may be associated with non-obstructive azoospermia.Fruitfly is a kind of model animal which has been used to study reproduction for a long time,regarding its short breeding cycle,gene conservation and the same reproductive process as human.We next used fruitfly as model system to screen surrounding genes within 10 kb of these SNPs,54 candidate genes were screened,and 7 of them were found to be associate with non-obstructive azzospermia.U2A/SNRPA1,a core component of major spliceosome,was among them.It was 4.5 kb downstream of the SNP rs7166401,which had the minimum P value.So we made a detailed analysis of this gene.We generated U2A loss of function mutant fruitfly by Crispr-Cas9.Phenotype analysis revealed that,in U2A mutant fruitfly testis,spermatogonia overproliferated.We can easily find spermatogonia cysts containing more than 16 cells per cyst.At the same time,the differentiation process of spermatogonia to spermatocytes were blocked.Only a small number of germ cells can differentiate into spermatocytes.While the number of germline stem cell was not significantly changed.These problems finally caused no mature sperm in U2A loss of function fruitfly seminal vesicle,which was very consistant with non-obstructive azoospermia.Next,we found that U2A took part in spermatogenesis by splicing mei-p26.In U2A loss of function mutant,last intron of mei-p26 was miss spliced,which caused Mei-p26 RNA and protein level severely decreased.What’s more,previous research told us that loss of Mei-p26 can lead to the same phenotype as U2A loss.So we defined mei-p26 as a target for U2A to attend male reproduction.Then we introduced U2A orthologous gene of human,SNRPA1 into mutant fruitfly,overexpression of SNRPA1 fully rescued the phenotype of U2A loss and mei-p26 miss splicing.This result revealed highly conservation of U2A/SNRPA1 between human beings and fruitfly.Previous research showed that U2A worked as a spliceosome factor by forming a dimer with U2B,so we paid attention to the amino acid of U2A,which interact directly with U2B.we purified proteins containing missense mutations of these amino acid,biochemical experiments demonstrated that,missense mutations in 15th,92nd and 148th amino acid of U2A disturbed the dimer to bind RNA,however,these missense mutant does not disturb the dimer to form.Choose U2A(Q148R)as an example,we overxpressed this missense mutation in wild type fruitfly,it interestingly showed donimant negative phenotype and mei-p26 miss splicing which just like U2A loss of function mutation.At last,we made a screen of other major spliceosome factors,in case to investigate whether they can also attend spermatogenesis in the same manner as U2A.Consequently,we found 6 of them may take part in male reproduction by splicing mei-p26.To conclude,our study showed that spliceosome complex may play a very important role during the process of male reproduction.Defects in spliceosome factors will lead severe problems of seprmatogenesis,and at last cause mature sperm loss,this mechanism is evolutionarily conserved.We hope our study will be helpful to look inside and to cure non-obstructive azoospermia in clinical.