Regulation of noncoding RNA biogenesis and function

非编码 RNA 生物合成和功能的调控

• 批准号: 8421393
• 负责人: Jeremy E Wilusz
• 金额: $ 9万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 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 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8421393
• 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; Libraries; 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; career development; human disease; in vivo; innovation; insight; next generation sequencing; novel; overexpression; programs; public health relevance; research study; skills; tissue/cell culture; tumor; tumorigenesis

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.

描述（由申请人提供）：大部分真核基因组被转录，产生复杂的转录物库，其中包括数万个单独的非编码RNA，具有很少或没有预测的蛋白质编码能力。其中包括研究充分的小RNA，如microRNA，以及许多其他类型的小而长的转录本，其功能和生物发生机制尚不清楚-但可能同样重要。MALAT 1基因座在许多人类癌症中过表达，并产生大量的长核保留非编码RNA。尽管是RNA聚合酶II转录物，但我们先前表明MALAT 1的3'末端不是通过典型切割/聚腺苷酸化产生的，而是通过RNA酶P识别和切割tRNA样结构产生的。这导致从MALAT 1基因座产生第二个非编码RNA，称为mascRNA，其是tRNA样的并输出到细胞质。mascRNA在进化上比长MALAT 1转录物明显更保守;然而，mascRNA的功能及其在癌症起始/进展中的作用尚未被探索。在Specific Aim 1中，我将使用一种新开发的表达质粒，该质粒重现了MALAT 1 3'末端加工，以在组织培养细胞中有效地过表达mascRNA。将鉴定通过调节mascRNA的表达诱导的基因表达和细胞表型的变化，从而揭示tRNA样小RNA在哺乳动物细胞中如何发挥功能的范例。在具体目标2中，我将描述尽管缺乏典型的poly（A）尾，但长MALAT 1转录物的3'端稳定的分子机制。这些实验将揭示新的见解多久不受切割/聚腺苷酸化的成绩单是耐降解和功能的基因表达。由于除了MALAT 1之外，很可能还有其他非编码RNA通过非经典机制在其3'端加工，因此下一代测序技术将用于特异性目标3，以特异性鉴定长poly（A）-RNA的3'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并耗尽丰富的管家RNA，例如核糖体RNA。然而，这一步骤也去除了所有缺乏poly（A）尾的长RNA，因此，大多数转录物经历了非典型的3'端加工机制。通过使用新的文库构建方法，这些先前“隐藏”的RNA的成熟3'末端将被揭示和表征，从而提供对可能控制RNA稳定性、定位或翻译效率的意想不到的调控机制的见解。在短期内，这种职业发展意识将使我能够在K99阶段大大扩展我的研究，进入新的，以前未探索的领域。夏普实验室和麻省理工学院的优秀培训环境不仅将大大促进指导研究，而且还赋予我过渡到独立学术教师职位的所有必要技能。从长远来看，我相信这些实验将为我自己的独立研究计划提供一个发展和繁荣的基础。总之，通过鉴定tRNA样小RNA的功能作用以及表征产生和稳定长RNA的非经典3'末端的机制，这些创新研究将揭示与人类癌症相关的非编码RNA的调控，功能和加工的关键新见解。