Specific Tandem 3UTR Patterns and Gene Expression Profiles in Mouse Thy1 Germline Stem CellsReport as inadecuate




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A recently developed strategy of sequencing alternative polyadenylation APA sites SAPAS with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells SSCs maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells GSCs, mainly referred to SSCs here, we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting MACS and then used the SAPAS method for analysis, using pluripotent embryonic stem cells ESCs and differentiated mouse embryonic fibroblast cells MEFs as controls. As a result, we obtained 99,944 polyA sites, approximately 40% of which were newly detected in our experiments. These polyA sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA lncRNA genes. We observed that GSCs tend to have shorter 3-UTR lengths while MEFs tend towards longer 3-UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3-UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal polyA switch in GSCs. Consistently APA reporter assay confirmed the total 3-UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal polyA site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.



Author: Yan Huang , Yuanyan Xiong , Zhuoheng Lin, Xuyang Feng, Xue Jiang, Zhou Songyang, Junjiu Huang

Source: http://plos.srce.hr/



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