EDITING OF THYMIDYLATE SYNTHASE RNA

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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.