Regulation of noncoding RNA biogenesis and function

非编码 RNA 生物合成和功能的调控

• 批准号: 8840774
• 负责人: Jeremy E Wilusz
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8840774
• 关键词: Affect; Area; Binding; Biogenesis; Cell Nucleus; Cell Proliferation; Cell physiology; Chromosomal translocation; Code; Complex; Conserved Sequence; Cytoplasm; Environment; Exonuclease; Faculty; Fishes; Foundations; Gene Expression; Gene Expression Profile; Generations; Genes; Genome; Housekeeping; Human; Human Genome; Individual; K-Series Research Career Programs; Libraries; MALAT1 gene; Malignant Neoplasms; Mammalian Cell; Mentors; Messenger RNA; Methionine; Methods; MicroRNAs; Molecular; Nuclear; Oncogenic; Open Reading Frames; Output; Phase; Phenotype; Plasmids; Poly A; Poly(A) Tail; Polyadenylation; Porifera; Positioning Attribute; Preparation; Process; Protein Biosynthesis; Proteins; RNA; RNA Polymerase II; RNA Stability; RNase P; Regulation; Research; Resistance; Ribosomal RNA; Role; Small RNA; Structure; System; Technology; Testing; Training; Transcript; Transfer RNA; Translations; Translocation Breakpoint; Untranslated RNA; cancer initiation; human disease; in vivo; innovation; insight; next generation sequencing; novel; overexpression; programs; research study; skills; tissue/cell culture; tumor; tumorigenesis

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.

项目概要/摘要 大多数真核基因组都被转录，产生复杂的转录本，其中包括 数以万计的单个非编码RNA，几乎没有或没有预测的蛋白质编码能力。之中 这些是经过充分研究的小RNA，例如microRNA，以及许多其他类别的小RNA和长RNA 转录物的功能和生物发生机制不太清楚，但可能同样重要。这 MALAT1 基因座在许多人类癌症中过度表达，并产生丰富的长核保留 非编码RNA。尽管是 RNA 聚合酶 II 转录本，但我们之前表明， MALAT1 不是通过典型的切割/聚腺苷酸化产生的，而是通过识别和切割 RNase P 产生的 tRNA 样结构。这导致从 MALAT1 生成第二个非编码 RNA 称为 mascRNA 的基因座，与 tRNA 类似并输出到细胞质。 masRNA明显更多 比长 MALAT1 转录本在进化上保守；然而，mascRNA 的功能及其在 癌症的发生/进展尚未被探索。在具体目标 1 中，我将使用新开发的 概括 MALAT1 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。通过调节表达引起的基因表达和细胞表型的变化 mascRNA 将被鉴定，从而为类似 tRNA 的小 RNA 在哺乳动物细胞中如何发挥作用提供范式 被揭露。在具体目标 2 中，我将描述长链 3' 末端的分子机制。 尽管没有典型的聚腺苷酸尾，MALAT1 转录物仍保持稳定。这些实验将揭示 关于不受切割/多聚腺苷酸化作用的转录本在多长时间内产生抗性的新见解 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 通过非规范机制在 3' 端进行处理的下一代测序技术将 可在特定目标 3 中用于特异性识别长 Poly(A) minus RNA 的 3' 末端。几乎所有以前的 表征转录组的研究使用了 Poly(A) 选择步骤来富集信使 RNA， 消耗丰富的看家 RNA，例如核糖体 RNA。然而，这一步也删除了所有长的 缺乏 Poly(A) 尾的 RNA，因此大多数转录物都经过非规范的 3' 末端处理 机制。通过使用新颖的文库构建方法，这些先前“隐藏”的成熟3'端 RNA 将被揭示和表征，提供对意想不到的调控机制的见解，这些机制可能 控制 RNA 稳定性、定位或翻译效率。短期内，该职业发展奖将 让我能够在 K99 阶段将我的研究极大地扩展到新的、以前未探索过的领域。这 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 但也赋予我过渡到独立学术教职职位所需的所有技能。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 研究项目可以成长和繁荣。总之，通过鉴定 tRNA 样小分子的功能作用 RNA 以及表征生成和稳定长链非规范 3' 末端的机制 RNA，这些创新研究将揭示有关 RNA 的调控、功能和加工的关键新见解 与人类癌症相关的非编码RNA。