MicroRNAs control mRNA fate by compartmentalizing mRNAs based on 3'UTR length

Introduction: we conduct comprehensive transcriptomic profiling analyses on three spermatogenic cell types (pachytene spermatocytes, round and elongating spermatids) purified from adult mouse testes using RNA-seq, and we determined not only the levels of both mRNAs and all known sncRNA species, but their cytoplasmic compartmentalization as well. Method: Pachytene spermatocytes, round and elongating/elongated spermatids were purified from adult mouse testes using the STA-PUT method. We fractionated the purified spermatogenic cells into RNP, monoribosome, and polyribosome fractions using a continuous sucrose gradient ultracentrifugation method, as described. Large RNAs were isolated using the AquaRNA RNA Purification Kit (Cat#5001MT, Mo Bi Tec, Inc.), whereas small RNAs were prepared by the mirPremier microRNA Isolation Kit (Cat#SNC10, Sigma). Constructions of mRNA and sncRNA libraries were conducted following our published protocols (Refs) and the next-gen sequencing was performed on a Hi-Seq 2000 sequencer with SE50 at the Genomics Microarray Core Facility of the UT Southwestern Medical Center (Dallas, TX). Result:Bioinformatics analyses revealed miRNAs were mostly enriched in RNPs and RNP-enriched miRNAs preferentially target RNP-enriched mRNAs. More interestingly, we found that miRNAs could distinguish shorter and longer 3'UTR transcript based on the distance between their binding sites and the stop codon. Conclusions:Overall, our genome-wide transcriptomic and bioinformatics analyses have revealed a highly likely mechanism through which miRNAs shape the haploid male germ cell-specific transcriptome characterized by RNP-enrichment of transcripts with shorter 3'UTRs. Pachytene spermatocytes, round and elongating/elongated spermatids were purified from adult mouse testes using the STA-PUT method. We fractionated the purified spermatogenic cells into RNP, monoribosome, and polyribosome fractions using a continuous sucrose gradient ultracentrifugation method, as described.引言：我们针对从成年小鼠睾丸中纯化得到的三种生精细胞类型（粗线期精母细胞、圆形精子细胞及延长型精子细胞）开展了全面的转录组表达谱分析，采用RNA测序（RNA-seq）技术。本研究不仅定量了信使RNA（mRNA）与所有已知小非编码RNA（small non-coding RNA, sncRNA）的表达水平，同时还分析了它们在细胞质中的区域化分布情况。 方法：我们通过STA-PUT法从成年小鼠睾丸中纯化得到粗线期精母细胞、圆形精子细胞及延长/伸长型精子细胞。如前文所述，我们采用连续蔗糖梯度超速离心法将纯化后的生精细胞分为核糖核蛋白复合物（ribonucleoprotein complex, RNP）、单核糖体及多核糖体组分。大RNA使用AquaRNA RNA纯化试剂盒（Cat#5001MT, Mo Bi Tec公司）进行提取，小RNA则通过mirPremier microRNA分离试剂盒（Cat#SNC10, Sigma公司）制备。mRNA与sncRNA文库的构建遵循我们已发表的实验方案（参考文献），下一代测序在德克萨斯大学西南医学中心（达拉斯，得克萨斯州）基因组芯片核心设施的Hi-Seq 2000测序仪上完成，采用单端50bp（SE50）测序模式。 结果：生物信息学分析显示，微小RNA（miRNA）主要富集于RNP组分中，且富集于RNP的miRNA倾向于靶向同样富集于RNP的mRNA。更为有趣的是，我们发现miRNA可根据其结合位点与终止密码子之间的距离，区分带有较短与较长3'非翻译区（3' untranslated region, 3'UTR）的转录本。 结论：总体而言，我们的全基因组转录组与生物信息学分析揭示了一套极具可能性的调控机制：miRNA可通过该机制塑造单倍体雄性生殖细胞特异性转录组，其特征为带有较短3'UTR的转录本在RNP中富集。粗线期精母细胞、圆形精子细胞及延长/伸长型精子细胞可通过STA-PUT法从成年小鼠睾丸中纯化得到。我们可采用连续蔗糖梯度超速离心法将纯化后的生精细胞分为RNP、单核糖体及多核糖体组分，如前文所述。