How cell stress and 3' end alterations control the metabolism of a cellular non-coding RNA

项目总结 RNA聚合酶II(POL II)典型地充当DNA依赖的RNA聚合酶(DdRP)，使用 双链DNA合成编码蛋白质的mRNAs和一些非编码的rna。Pol II还拥有 RNA依赖的RNA聚合酶活性(RDRP)，它使用RNA作为模板来合成RNA。一个 Pol II RdRP活性的例子是非编码B2 RNA的3‘端延伸以产生延伸的 B2(EB2)RNA。B2RNA是由短的散布元素(Sine)编码的，存在于超过350,000个 由于逆转录转座而在小鼠基因组中复制。将这些元素随机插入基因组 可能是有害的，这取决于哪个区域或基因被破坏，在某些情况下可能会导致疾病。 在细胞应激时，来自B2Sine的非编码B2RNAs的转录大大增加，从而 增加了B2Sine逆转位的可能性。Pol II的RdRP活性可控制上述水平 通过产生eB2 RNA来促进其降解，从而在转录后对B2RNA产生影响。拟议的工作将 鉴定细胞中所有形式的3‘修饰的B2 RNA，测量它们的稳定性，并确定3’末端延伸的方式 B2RNA改变了其在细胞内的定位和逆转录转座。 B2RNA与Pol II结合，全局抑制转录，并经历Pol II依赖的18 核苷酸RdRP延伸形成eB2 RNA。此外，eB2RNA的半衰期大大缩短，因为 与B2RNA相比。从B2RNA形成eB2RNA被认为是一种自我调节机制 克服转录抑制，促进eB2RNA的解离和降解。到目前为止， B2RNA的大部分特征都是通过生化实验进行的，而这些实验并没有 涵盖了细胞系统的复杂性，可能会改变Pol II RdRP活性的效果。这 该提案旨在了解RNA聚合酶如何在转录后控制RNA新陈代谢。 以确定细胞3‘修饰的B2 RNA物种由于延伸，加工，并确定如何应激 影响这些物种，将开发一种B2RNA特异性测序方案(B2RNA-seq)。要量化 B2RNA物种的稳定性，B2RNA-seq将偶联到溴代吡啶脉冲追踪剂上。此外，要 观察应激前后B2和eB2RNA在细胞内的定位，RNA荧光原位观察 将利用杂交(FISH)。FISH将与免疫荧光实验相结合，以确定 RNA和潜在的加工机制之间的共址。此外，要确定3‘扩展如何 而细胞应激影响B2RNA的逆转录转座能力，逆转录转座的细胞培养模型将 被利用。总之，这些研究将提供对B2 RNA表达之间的相互作用的理解， Pol II RdRP延伸、RdRP调节的降解、细胞定位和细胞应激对其生命周期的影响 这个ncRNA。

PROJECT SUMMARY RNA Polymerase II (Pol II) canonically acts as a DNA-dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein-encoding mRNAs and some non-coding RNAs. Pol II also has RNA-dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3’ end extension of the non-coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA is encoded by Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Random insertion of these elements into the genome could be deleterious, depending on which region or gene is disrupted, and in some cases can cause disease. Upon cellular stress, transcription of non-coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post-transcriptionally by generating eB2 RNA to promote its degradation. The proposed work will identify all forms of 3’ modified B2 RNAs in cells, measure their stabilities, and determine how 3’ end extension of B2 RNA changes its intracellular localization and retrotransposition. B2 RNA binds to Pol II, globally represses transcription, and undergoes a Pol II-dependent 18 nucleotide RdRP extension to form eB2 RNA. Furthermore, eB2 RNA has a drastically reduced half-life as compared to B2 RNA. The formation of eB2 RNA from B2 RNA is thought to be an autoregulatory mechanism to overcome transcriptional repression and promote the dissociation and degradation of eB2 RNA. To date, the majority of the characterization of B2 RNA has been through biochemical experiments, which do not encompass the complexity of a cellular system that could alter the efficacy of Pol II RdRP activity. This proposal aims to understand how an RNA polymerase controls RNA metabolism post-transcriptionally. To identify cellular 3’ modified B2 RNA species due to extension, processing, and determine how stress affects these species, a B2 RNA-specific sequencing scheme (B2 RNA-seq) will be developed. To quantify the stabilities of B2 RNA species, B2 RNA-seq will be coupled to bromouridine pulse-chase. Furthermore, to observe the localization of B2 and eB2 RNAs in cells before and after stress, RNA Fluorescence in situ hybridization (FISH) will be utilized. FISH will be coupled to immunofluorescence experiments to determine colocalization between RNAs and potential processing machinery. Additionally, to determine how 3’ extension and cellular stress affect the ability of B2 RNA to retrotranspose, a cell-culture model of retrotransposition will be used. Altogether, these studies will provide an understanding of the interplay between B2 RNA expression, Pol II RdRP extension, RdRP-regulated degradation, cellular localization, and cellular stress on the life cycle of this ncRNA.