鸡的小RNA文库构建和非编码RNA的规模筛选

【作者】 黄守均；

【导师】 朱大海；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 非编码RNA在生命活动中起着非常重要的作用,如参与染色质结构的调节,转录和转录后加工的调控,蛋白质合成、转运和活性的调控等,但是已知的非编码RNA只占生物体内全部非编码RNA的很少一部分,绝大多数的非编码RNA还有待于被发现和进行功能研究。通过构建特殊的cDNA文库发现新的非编码RNA是一个比较有效的方法。本研究以鸡为模式生物,构建了富集小分子非编码RNA的文库,从中找到一大批新的非编码RNA,并利用Northern杂交实验验证了这些非编码RNA的存在。对这些新的非编码RNA进行的整体性的研究使我们对非编码RNA有了更深入的了解。首先,通过分析各种非编码RNA在鸡的不同组织和骨骼肌不同发育阶段中的表达,发现绝大多数非编码RNA在时空上具有不同的表达水平,说明这些非编码RNA在体内的产生和降解受到了严格的调控,提示它们在某些生命活动中具有重要的作用;同时发现鸡中许多的非编码RNA在小鼠和人类中并没有同源RNA分子的存在,基因组水平的比对也不能找到同源的序列,这说明生物体在进化中会产生相当多的物种特有的非编码RNA,这与蛋白质编码基因所具有的保守性相比具有显著的不同,这些非编码RNA可能在很大程度上决定了物种的独特性。本实验还发现了一个只在鸡的骨骼肌和心肌中表达的非编码RNA——MuN121,Blast分析表明它定位于一个已知的蛋白质编码基因的内含子之中,Northern杂交结果显示MuN121和该宿主基因在RNA水平具有一致的表达谱。为了研究MuN121的产生是经独立转录而来还是从pre-mRNA加工而来,我们构建了野生型和该内含子5’端剪接供体位点突变的2种载体,转染293T细胞发现突变体不能产生ncRNA而野生型能产生。表明MuN121的产生依赖于pre-mRNA的成功剪接,MuN121和宿主基因受相同的转录机制的调控。更多还原

【Abstract】 In the last several years, noncoding RNAs (ncRNAs) have been identified in cells of all three kingdoms. It has been identified that ncRNAs can regulate gene regulation at different levels, such as chromatin modification; transcriptional and post-transcriptional regulation; protein synthesis, translocation and activity controlling. However, there is still a large number of ncRNAs need to be identified. Here we use chicken as a model to construct an ncRNA enriched cDNA library and then systematicly study ncRNAs’ expression, conservation and biological function. We found that expression of the majority of ncRNAs is spatio-temporal regulated, suggestting that these ncRNAs play important roles during development. RNA blotting and comparative genomics analysis found that many ncRNAs do not have homologue in mouse and human. These results indicate that ncRNAs may contribute largely to the specialties of cell lineage.We also found a muscle specific expressed ncRNA, MuN121. Blast analysis found that it is encoded in the intron of a host gene. MuN121 and The host gene have identical expression pattern, we also found that MuN121 biogenesis is depend on successful splicing of the encoded intron. These results suggest that MuN121 is produced from the pre-mRNA of the host gene. MuN121 may play an important role in host gene regulation or in regulating the same pathway that the host gene is involved.更多还原