EDITING OF THYMIDYLATE SYNTHASE RNA

胸腺苷酸合成酶是负责胸腺苷酸从头合成的酶，是化疗中的主要靶点。胸苷酸基因表达的调控似乎发生在几个水平上，主要影响是转录后的。这个实验室发现了一种基因（rTS），它可以产生一种自然发生的反义RNA来对抗TS RNA。该基因的表达模式（随着培养细胞密度的增加，表达水平更高）表明与TS基因表达有关。转染实验支持了这些基因之间的关系，其中rTSalpha （TS基因的天然反义RNA转录物）可以下调TS mRNA和TS蛋白水平，并抑制细胞生长。利用RT/PCR（逆转录/PCR），我们发现H630细胞（人结肠癌）中TS pre-mRNA在与rTSalpha互补的区域被编辑。编辑过程可能涉及在TS前mRNA的五个特定位点将腺苷转化为肌苷（即腺苷脱氨）。基于对其他RNA的编辑描述，预计这一过程是通过rTSalpha RNA与TS前mrna的杂交介导的，然后由作用于RNA的腺苷脱氨酶（ADARs）介导腺苷脱氨。初步数据表明，编辑可能在TS前mRNA从成熟过程中移除，从而导致TS mRNA的丢失中发挥作用。rTSalpha RNA的反义区似乎足以诱导TS mRNA的丢失。我们拟在四个特定目的中研究TS前mRNA编辑与TS基因表达调控和实验性化疗的机制和相关性。确定TS pre-mRNA RT/PCR产物中显示A到G转换的5个位点是否由腺苷脱胺引起，并建立一种定量TS pre-mRNA编辑的方法。测定rTSalpha RNA和TS mRNA是否原位形成双链。2. 评估TS pre-mRNA编辑是否随生长和/或细胞周期变化。3. 确定rTSalpha RNA是否可以调节TS pre-mRNA编辑水平和相应的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. 生成rtsalpha -反义阳痿细胞系，研究减毒或消除反义RNA对TS基因表达和药物敏感性调控的影响。

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.