Regulation of noncoding RNA biogenesis and function

项目总结/摘要 大部分真核基因组被转录，产生复杂的转录物库，包括 数以万计的个体非编码RNA具有很少或没有预测的蛋白质编码能力。之间 这些都是研究得很好的小RNA，如microRNA，以及许多其他类型的小而长的RNA。 转录本的功能和生物发生机制尚不清楚，但可能同样重要。的 MALAT 1基因座在许多人类癌症中过表达，并产生丰富的长核保留的 非编码RNA。尽管是RNA聚合酶II的转录本，我们以前表明， MALAT 1不是通过典型的切割/多聚腺苷酸化产生的，而是通过识别和切割一个 这导致从MALAT 1产生第二个非编码RNA。 一种称为mascRNA的基因座，它是tRNA样的并输出到细胞质。mascRNA明显比 在进化上比长MALAT 1转录本保守;然而，mascRNA的功能及其在 癌症的发生/发展尚未被探索。在具体目标1中，我将使用新开发的 再现MALAT 1 3'末端加工以在组织中有效过表达mascRNA的表达质粒 培养细胞。通过调节基因表达诱导的基因表达和细胞表型的变化 mascRNA将被鉴定，从而为tRNA样小RNA在哺乳动物细胞中的功能提供范例， 被揭露。在具体目标2中，我将描述长链的3'末端的分子机制。 MALAT 1转录物是稳定的，尽管缺乏典型的poly（A）尾。这些实验将揭示 新的见解，多长时间不受切割/多聚腺苷酸化的转录本， 降解和基因表达的功能。因为除了MALAT 1外，很可能还有其他非编码RNA 通过非规范机制在其3'端加工，下一代测序技术将 在特异性目标3中用于特异性鉴定长poly（A）-RNA的3'末端。几乎所有以前的 表征转录组的研究已经使用聚（A）选择步骤来富集信使RNA， 消耗大量的管家RNA，如核糖体RNA。但是，此步骤也会删除所有长 缺少poly（A）尾的RNA，因此，大多数转录物经历非典型的3'末端加工 机制等通过使用一种新的文库构建方法， RNA将被揭示和表征，提供对意想不到的调控机制的见解， 控制RNA稳定性、定位或翻译效率。在短期内，这一职业发展奖将 让我大大扩展我的研究到新的，以前未开发的领域在K99阶段。的 夏普实验室和麻省理工学院的优秀培训环境不仅将极大地促进指导研究， 同时也赋予我所有必要的技能，使我能够过渡到一个独立的学术教师职位。在 从长远来看，我相信这些实验将为我自己的独立研究奠定基础。 研究项目可以蓬勃发展。总之，通过鉴定tRNA样小分子的功能作用， RNA以及表征产生和稳定长RNA的非典型3'末端的机制。 这些创新性的研究将揭示关键的新见解的调控，功能和加工， 与人类癌症相关的非编码RNA。

PROJECT SUMMARY/ABSTRACT Most of the eukaryotic genome is transcribed, yielding a complex repertoire of transcripts that includes tens of thousands of individual noncoding RNAs with little or no predicted protein-coding capacity. Among these are well-studied small RNAs, such as microRNAs, as well as many other classes of small and long transcripts whose functions and mechanisms of biogenesis are less clear - but likely no less important. The MALAT1 locus is over-expressed in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being an RNA polymerase II transcript, we previously showed that the 3' end of MALAT1 is not produced by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. This results in the generation of a second noncoding RNA from the MALAT1 locus known as mascRNA that is tRNA-like and exported to the cytoplasm. mascRNA is significantly more evolutionarily conserved than the long MALAT1 transcript; however, the function of mascRNA and its role in cancer initiation/progression have not been explored. In Specific Aim 1, I will use a newly developed expression plasmid that recapitulates MALAT1 3' end processing to efficiently overexpress mascRNA in tissue culture cells. Changes in gene expression and cellular phenotype induced by modulating the expression of mascRNA will be identified, allowing paradigms for how tRNA-like small RNAs function in mammalian cells to be revealed. In Specific Aim 2, I will characterize the molecular mechanisms by which the 3' end of the long MALAT1 transcript is stabilized despite the absence of a canonical poly(A) tail. These experiments will reveal new insights into how long transcripts not subjected to cleavage/polyadenylation are made resistant to degradation and function in gene expression. As there are very likely other noncoding RNAs besides MALAT1 that are processed at their 3' ends via non-canonical mechanisms, next-generation sequencing technology will be used in Specific Aim 3 to specifically identify the 3' ends of long poly(A) minus RNAs. Nearly all previous studies characterizing the transcriptome have used a poly(A) selection step to enrich for messenger RNAs and deplete abundant housekeeping RNAs, such as ribosomal RNAs. However, this step also removes all long RNAs that lack poly(A) tails and, therefore, most transcripts subjected to non-canonical 3' end processing mechanisms. By using a novel library construction method, the mature 3' ends of these previously "hidden" RNAs will be revealed and characterized, providing insights into unexpected regulatory mechanisms that may control RNA stability, localization, or translation efficiency. In the short term, this career development award will allow me to greatly expand my research into new, previously unexplored areas during the K99 phase. The excellent training environment in the Sharp lab and MIT will greatly facilitate not only the mentored research but also endow me with all the necessary skills to transition to an independent academic faculty position. In the long term, I am confident that these experiments will provide a foundation on which my own independent research program can grow and flourish. In summary, by identifying the functional role of tRNA-like small RNAs as well as characterizing the mechanisms that generate and stabilize non-canonical 3' ends of long RNAs, these innovative studies will reveal key new insights into the regulation, functions, and processing of noncoding RNAs that are relevant in human cancer.