Proteomics Study Of Small Proteins Derived From Mouse Testis Non-coding Genes

Spermatogenesis is a very complicated process,including spermatogonial stem cell self-renewal and differentiation,spermatocyte meiosis and sperm cell deformation process,finally from circular sperm cells mature into a tadpole shape.Due to the complex process of spermatogenesis molecular regulation,the research of spermatogenesis in the final executor of life activities of protein level is particularly important.Using proteomics methods,we ever identified 7346 and 4675 kinds of proteins from adult testicular tissue and mature sperm,respectively.These identified proteins showed that the protein regulation during spermatogenesis is very complicated.Proteomics research,however,should be based on known protein sequence databases for annotation protein sequences.At present,the existing method for protein coding genes is insufficient.Surveys found that non-coding regions and those regions coding large molecular weight protein gene lurk an undiscovered short reading frames which may have a short protein coding functional potential.In addition,limited by technical method,within the scope of the genome of mass prediction protein-coding genes,generally only consider the length not less than 100 amino acid composition of protein.Moreover,there is the difficulty of finding a short protein method only using biological information.With the continuous development of technology of mass spectrometry and proteomics technology,the ability to identify low abundance proteins also increased.There are some studies combinations of proteomics,genomics and bioinformatics methods of high-throughput screening,to identify and validate short protein.In addition,the traditional views consider that lncRNA does not code for proteins,but which may be involved in the epigenetic regulation of cell surface through a variety of ways.The functional study of lncRNA in the testis is little.For RNA sequencing,researches shows that about 33% of the genes in the long non-coding gene can be expressed in testis specifically.So lncRNA might be an important mechanism for the regulation of spermatogenesis.New research shows that part of lncRNA maybe encode short proteins,as a result,looking for possible coding short reading frames and study on the expression and expression in testis function in lncRNA,could provide a new idea for elucidating the regulatory mechanisms of lncRNA and the regulation of spermatogenesis.With a 30 kD molecular weight filter membrane filtering,and combined with the strategy of proteomics and bioinformatics,we have been preliminary identify 57 new TlncSEPs(Testicular LncRNA short ORFs encoded proteins).RT-PCR and Real-Time PCR detect the expression of these genes.In this new appraisement of testicular short proteins there are three short proteins from the typical LncRNA;about forty short proteins encoded by mRNA-like LncRNA,such as TlncSEP N343248 and TlncSEP N277827.What's more,fourteen short proteins from traditional pseudogenes.Through bioinformatics analysis,we found that these short proteins may be involved in the nuclear DNA binding,collection of motor proteins,metal ions,and energy metabolism and turn into the way such as signal,suggesting that the complexity of mice testis proteins and functional diversity.Besides,these small proteins associated with the subcellular localization of small protein is distributed in the cytoplasm,cell nucleus and cytoplasm vacuoles.And some other subcellular structures such as the ribosome or mitochondria.Small molecular biology functional proteins may be involved in the nuclear DNA binding,motor protein collection,metal ions,and energy metabolism.At last,Homologous sequence alignment found fifteen new species identification of short proteins are highly conservative in anthropogenic.Furthermore,we have randomly selected some genes to analysis the expression of non-coding gene and gene transcription at the m RNA level.On the one hand,the RT-PCR experimental results show that the expression of non-coding gene in testicular tissue was verified in the actual reliability of transcription level of our group.On the other hand,the real-time PCR experimental results show that encoding genes such as: TlncSEP N269302,TlncSEP N343248,TlncSEP N277827 whose expression quantity increased gradually,especially the expression quantity increased significantly between 1w to 5w.In summary,the non-coding regions hide undiscovered short reading frame maybe code functional short proteins.Because of a large number of higher expressions of lncRNA,and with a complex translation regulation of testicular tissue,short proteins may be form important regulatory mechanism.Therefore,it is necessary to understand the construction of mouse testis lncRNA expression spectrum and to discovery and identify short proteins related to spermatogenesis.For our further study,it will be great significance to clarify the role and the regulation mechanism of short proteins in spermatogenesis.