Regulation of noncoding RNA biogenesis and function

描述(申请人提供)：真核基因组的大部分是转录的，产生了一个复杂的转录库，其中包括数万个独立的非编码RNA，几乎没有预测的蛋白质编码能力。其中包括被充分研究的小RNA，如microRNAs，以及许多其他类别的小和长转录本，其生物发生的功能和机制不太清楚，但可能同样重要。MALAT1基因座在许多人类癌症中过度表达，并产生丰富的长核滞留的非编码RNA。尽管MALAT1是RNA聚合酶II的转录本，但我们先前证明MALAT1的3‘端不是通过典型的切割/多聚腺苷基化产生的，而是通过RNaseP识别和切割tRNA样结构而产生的。这导致从MALAT1基因座产生第二个非编码RNA，称为mascRNA，它是tRNA样并输出到细胞质。MascRNA在进化上明显比长的MALAT1转录本保守；然而，mascRNA的功能及其在癌症发生/发展中的作用尚未被探索。在特定的目标1中，我将使用一种新开发的表达载体，它概括了MALAT1 3的末端处理，以便在组织培养细胞中高效地过表达mascRNA。将识别通过调节mascRNA的表达而引起的基因表达和细胞表型的变化，从而揭示tRNA样小RNA如何在哺乳动物细胞中发挥作用的范例。在特定的目标2中，我将描述分子机制，通过这些机制，长MALAT1转录本的3‘端在缺乏典型的聚(A)尾巴的情况下得到稳定。这些实验将揭示新的见解，即不受切割/多聚腺苷作用的转录本能在多长时间内抵抗降解，并在基因表达中发挥作用。由于除MALAT1外，很可能还有其他非编码RNA在其3‘端通过非规范机制进行处理，因此下一代测序技术将被用于特定的目标3，以专门识别长的聚(A)-RNA的3’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，并耗尽丰富的管家RNA，如核糖体RNA。然而，这一步骤也移除了所有缺乏Poly(A)尾巴的长RNA，因此，大多数转录本受到非规范的3‘端处理机制的影响。通过使用一种新的文库构建方法，这些以前“隐藏”的RNA的成熟3‘端将被揭示和表征，从而为控制RNA稳定性、本地化或翻译效率的意想不到的调控机制提供见解。在短期内，这一职业发展AWAR将使我能够在K99阶段将我的研究极大地扩展到以前未探索的新领域。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，还将赋予我过渡到独立学术教员职位所需的所有技能。从长远来看，我相信这些实验将为我自己的独立研究项目的发展和繁荣提供一个基础。总之，通过确定tRNA样小RNA的功能作用以及表征产生和稳定长RNA非规范3‘端的机制，这些创新研究将揭示与人类癌症相关的非编码RNA的调节、功能和处理的关键新见解。

DESCRIPTION (provided by applicant): 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 awar 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.