The Function Study Of Mouse BOULE? Protein And Boule Circular RNA In Spermatogenesis

mRNA-dependent protein synthesis is crucial to gene expression and cellular function in eukaryotic cells.Translation generally starts with AUG codon in eukaryotes,while occasional translation with non-AUG inhiation codon have been attributed to promiscuous translation and was considered an exception.Recently,with the advance of ribosome profiling technology,non-AUG initiation translation have been found in the 5'-UTR(untranslated region)of many eukaryotic genes.This phenomenon was more frequent in eukaryotic cells upon experiencing stress or in cancer cells,indicating that the non-AUG initiation event was not simply translation errors,but the biological significance of non-AUG initiation translation remains undetermined and no animal model lacking non-AUG protein was reported.Boule gene,conserved in almost all animal species from fly to human,has a crucial regulatory function in spermatogenesis.In this study,we identified novel BOULE isoforms without canonical AUG start codon,designated BOULE?s.BOULE?s translation is initiated from an AUU codon in Boule exon1 d and an evolutionarily conserved hairpin structure downstream of AUU is crucial for this noncanonical AUU initiation.However,BOULE?s knockout mice lacking all BOULE?s were fertile,indicating that BOULE?s are dispensable for mouse spermatogenesis.Circular RNAs(circ RNAs),a new member of nc RNA(non-coding RNA)family,are covalently closed from single-stranded RNA transcripts.Although circ RNAs have been discovered more than 40 years ago,they for long time were thought to be byproducts of abnormal splicing of pre-m RNA with little function.However,recent studies showed that the expression of circ RNAs were widespread among almost all eukaryotes.Circ RNAs are generally derived from back-splicing of pre-m RNA transcripts,resulting in a circular structure with a 3?,5?-phosphodiester bond at junction site.In eukaryotes,biogenesis of circ RNAs depends on spliceosome-dependent backsplicing,reverse complementary sequences in flanking introns,and RBPs(RNA binding proteins).Recently,studies have revealed that some circ RNAs may have potential cellular functions through sponging micro RNA,promoting transcription,coding for short peptides,regulating the process of cell cycle and ribosomal RNA maturation,and involving in the development of diseases.However,the biological function of circ RNAs remain largely unclear,especially in spermatogenesis.In this study,we detected exonic circ RNAs generated from a key reproductive Boule gene,and these Boule circ RNAs are conserved from fly to human and show an expression pattern specific to or predominate in the testis.We successfully constructed fly bol circ RNAs knockout model via removing introns of bol gene coding region.The ability to maintain long-term fertility was significantly reduced in male flies lacking bol circ RNAs.Furthermore,this subfertility phenotype was more severe when the flies were reared at higher temperature.Mature sperm was significantly reduced in number,with elevated apoptosis and increased number of abnormal sperm in seminal vesicle.In summary,we discovered highly conserved circ RNAs from the orthologous locus of Boule from fly to human and showed flies lacking those bol circ RNAs have reduced fecundity,demonstrating a critical requirement in maintaining long-term fertility.