Regulation of noncoding RNA biogenesis and function

项目摘要/摘要 大多数真核生物的基因组都是转录的，产生了一系列复杂的转录本，包括 数以万计的个体非编码RNA几乎没有或几乎没有预测的蛋白质编码能力。其中 这些都是研究得很好的小RNA，如microRNAs，以及许多其他类别的小和长的 生物发生的功能和机制不太清楚，但可能同样重要的转录本。这个 MALAT1基因座在许多人类癌症中过度表达，并产生丰富的长时间滞留的核 非编码RNA。尽管是RNA聚合酶II的转录本，我们之前已经证明了 MALAT1不是通过典型的切割/多聚腺苷基化产生的，而是通过识别和切割一种 RNase P.的tRNA样结构。这导致从MALAT1产生第二个非编码RNA 被称为mascRNA的类似tRNA并输出到细胞质的基因座。MascRNA明显更多 在进化上比长的MALAT1转录本保守；然而，mascRNA的功能及其在 癌症的发生/发展还没有被研究过。在具体目标1中，我将使用一种新开发的 重述MALAT1 3‘末端处理以在组织中高效过表达mascRNA的表达载体 培养细胞。基因表达调控对细胞表型和基因表达的影响 MascRNA将被识别，从而允许研究类似tRNA的小RNA在哺乳动物细胞中如何发挥作用 会被揭穿。在具体目标2中，我将描述长链3‘端的分子机制 MALAT1转录本是稳定的，尽管没有典型的聚(A)尾巴。这些实验将揭示 对不受切割/多聚腺苷酸化作用的转录体产生多长时间抗性的新见解 基因表达中的降解和功能。因为除了MALAT1之外，很可能还有其他非编码RNA 在它们的3‘端通过非规范机制进行处理，下一代测序技术将 在特定目的3中使用，以特定识别长聚(A)减去RNA的3‘末端。几乎所有以前的 对转录组的研究使用了聚(A)选择步骤来丰富信使RNA和 消耗大量的管家RNA，如核糖体RNA。然而，这一步也删除了所有长的 缺少Poly(A)尾的RNA，因此大多数转录本都经历了非规范的3‘端处理 机械装置。通过使用一种新的文库构建方法，这些成熟的3‘端以前被“隐藏” RNA将被揭示和表征，提供对可能 控制RNA的稳定性、本地化或翻译效率。在短期内，这个职业发展奖将 请允许我在K99阶段将我的研究极大地扩展到以前未探索的新领域。这个 夏普实验室和麻省理工学院良好的培训环境不仅将极大地促进指导研究 但这也赋予了我所有必要的技能，让我能够过渡到独立的学术教职。在 从长远来看，我相信这些实验将为我自己的独立提供一个基础 研究项目可以成长和蓬勃发展。总之，通过确定tRNA样小分子的功能作用 以及描述产生和稳定Long的非规范3‘端的机制 RNA，这些创新的研究将揭示对调节、功能和加工的关键新见解 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.