EDITING OF THYMIDYLATE SYNTHASE RNA

胸苷酸合成酶 RNA 的编辑

• 批准号: 6150367
• 负责人: BRUCE JEFFREY DOLNICK
• 金额: $ 20.48万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-06 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6150367
• 关键词: adenosine; amination; antisense nucleic acid; cell cycle; chemosensitizing agent; enzyme activity; gene expression; genetic regulation; messenger RNA; polymerase chain reaction; posttranscriptional RNA processing; protein structure; thymidylate synthase; tissue /cell culture

Thymidylate synthase is the enzyme responsible for the de novo synthesis of thymidylate and is a major target in chemotherapy. Regulation of thymidylate gene expression appears to occur at several levels, with the major effects being post-transcriptional. This laboratory discovered a gene (rTS) that generates a naturally occurring antisense RNA to TS RNA. The expression pattern of this gene (higher levels of expression as cultured cell density increases) suggests a relationship to TS gene expression. A relationship between these genes is supported by transfection experiments where rTSalpha (a natural antisense RNA transcript to the TS gene) can be shown to down-regulate TS mRNA, TS protein levels, and inhibit cell growth. Using RT/PCR (reverse transcription/PCR) we have now discovered TS pre-mRNA is edited in H630 cells (human colon cancer) in the region of complementarity with rTSalpha. The editing process likely involves conversion of adenosine to inosine (i.e. adenosine deamination) at five specific sites in the TS pre- mRNA. Based upon the editing described for other RNAs, it is expected this process is mediated by the hybridization of rTSalpha RNA to TS pre-mRNA, followed by adenosine deamination by adenosine deaminase(s) that act on RNA (ADARs). Preliminary data suggest editing may play a role in the removal of TS pre-mRNA from the maturation process leading to a loss of TS mRNA. The antisense region of rTSalpha RNA appears to be sufficient to induce the loss of TS mRNA. We propose to investigate the mechanism and relevance of TS pre- mRNA editing to the regulation of TS gene expression and experimental chemotherapy in four Specific Aims 1. Determining whether the five sites displaying A to G transitions in RT/PCR products from TS pre-mRNA result from adenosine deamination and developing a method to quantitate TS pre-mRNA editing. Determine whether rTSalpha RNA and TS mRNA form duplexes in situ. 2. Evaluate whether TS pre-mRNA editing changes with growth and/or cell cycle. 3. Determine whether rTSalpha RNA can modulate the level of TS pre-mRNA editing and consequentially TS protein levels, activity and sensitivity to TS inhibitors, and examine the kinetics of these phenomena. 4. Generate rTSalpha-antisense impotent cell lines and study the effect of attenuating or eliminating antisense RNA on regulation of TS gene expression and drug sensitivity.

胸苷酸合酶是负责从头合成胸苷酸的酶，是化学疗法的主要靶标。胸苷酸盐基因表达的调节似乎在几个水平上发生，主要影响是转录后的。 该实验室发现了一个基因（RTS），该基因对TS RNA产生天然存在的反义RNA。该基因的表达模式（随着培养的细胞密度增加，表达水平较高）表明与TS基因表达有关系。 这些基因之间的关系得到了转染实验的支持，即可以证明RTSALPHA（自然反义RNA转录到TS基因）下调TS mRNA，TS蛋白水平和抑制细胞生长。 使用RT/PCR（逆转录/PCR），我们现在发现TS Pre-MRNA在与RTSalpha互补区域的H630细胞（人结肠癌）中进行了编辑。 编辑过程可能涉及将腺苷转化为TS前mRNA的五个特定部位的腺苷（即腺苷脱氨酸）。 基于针对其他RNA所述的编辑，预计该过程是由RTSalpha RNA与TS Pre-MRNA的杂交介导的，然后是作用于RNA（ADARS）的腺苷脱氨酸酶的腺苷脱氨酸。 初步数据表明，编辑可能在从成熟过程中去除TS前MRNA中起作用，从而导致TS mRNA损失。 RTSalpha RNA的反义区似乎足以诱导TS mRNA的丧失。我们建议研究在四个特定目标中Ts Pre-mRNA编辑对TS基因表达和实验化学疗法调节的机制和相关性1。确定是否确定来自TS PRE-MRNA中a腺苷脱氨酸引起的RT/PCR产物中的五个位点是由腺苷脱氨酸引起的，并开发了量化TS Pre-MRNA的方法。 确定RTSALPHA RNA和TS mRNA是否原位形成双链体。 2。评估TS前MRNA编辑是否随生长和/或细胞周期而变化。 3。确定RTSALPHA RNA是否可以调节TS前MRNA编辑水平，并导致TS蛋白水平，活性和对TS抑制剂的敏感性，并检查这些现象的动力学。 4。产生RTSALPHA抗药性的无能细胞系，并研究衰减或消除反义RNA对调节TS基因表达和药物敏感性的影响。