Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells.
Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells.
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DOI：
10.1371/journal.pone.0145417
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Huang J
中科院分区：
文献类型：
--
作者：
Huang Y;Xiong Y;Lin Z;Feng X;Jiang X;Songyang Z;Huang J
最近开发的一种利用第二代测序技术对选择性多聚腺苷化（APA）位点（SAPAS）进行测序的策略，可用于探索串联APA位点的全基因组模式和全球基因表达谱。精原干细胞（ssc）在雄性哺乳动物中维持长期的生殖能力。ssc自我更新和产生成熟精子的详细机制尚不清楚。为了了解雄性生殖系干细胞（GSCs，这里主要是SSCs）的特异性选择性多腺苷化模式和全局基因表达谱，我们采用磁激活细胞分选（MACS）方法从小鼠睾丸中分离纯化了Thy1+细胞，然后使用SAPAS方法进行分析，以多能胚胎干细胞（ESCs）和分化的小鼠胚胎成纤维细胞（MEFs）为对照。结果，我们获得了99,944个聚(a)位点，其中约40%是在我们的实验中新检测到的。这些多聚(A)位点来自三种小鼠细胞类型，覆盖了17,499个基因，其中包括831个长链非编码RNA （lncRNA）基因。我们观察到GSCs趋向于具有较短的3'UTR长度，而mef趋向于具有较长的3'UTR长度。我们还鉴定出1337个在GSCs中高表达的基因，这些基因与GSCs的功能特征高度一致。通过详细的生物信息学分析，我们在GSCs中发现了3' utr上的APA位点转换事件和许多新的特异性表达基因，我们通过实验证实了这一点。此外，采用qRT-PCR验证了GSCs中334个具有远端到近端poly(A)开关的基因的几个事件。与mef相比，一致的APA报告基因实验证实GSCs中总3'UTR缩短。我们还分析了GSCs中优先使用的近端聚(A)位点周围的顺式元素，发现富c元素可能有助于这种调节。总的来说，我们的研究结果确定了表达水平和聚腺苷化位点谱，这些数据为GSC生命周期和精子发生的潜在过程提供了新的见解。
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 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) 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 poly(A) 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 poly(A) 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.