IDENTIFICATION OF RAPID TURNOVER TRANSCRIPTS OVEREXPRESSED IN THYROID-TUMORS AND THYROID-CANCER CELL-LINES - USE OF A TARGETED DIFFERENTIALRNA DISPLAY METHOD TO SELECT FOR MESSENGER-RNA SUBSETS

The mRNAs of transiently expressed proteins such as cytokines and prot o-oncogenes are commonly subject to rapid transcriptional activation a nd degradation, Transcript turnover is determined in part by associati on of certain proteins with consensus AU-rich motifs (AUUUA) in the 3' -untranslated region of the transcripts, Here we report a modification of differential RNA display (DRD) to detect differentially expressed rapid turnover mRNAs containing AU-rich motifs from thyroid cancer tis sues and cell lines, RNA of normal and thyroid cancer tissues was diff erentially displayed using a 3' anchor primer to the poly(A) tail and an arbitrary 5' primer incorporating an AUUUA sequence, The appropriat eness of the strategy was established by its ability to display known early response genes, such as c-fos, using partially degenerate primer s. To test whether the novel cDNAs isolated coded for transcripts subj ect to rapid turnover, they were used as probes for Northern blots of RNA from clonal human thyroid carcinoma cell lines treated for varying periods with either cycloheximide or actinomycin D, A number of novel differentially expressed cDNA fragments were isolated from human papi llary thyroid carcinoma tissues, among them a cDNA with zinc finger mo tifs and homology to other zinc finger proteins, Using this fragment t o probe a cDNA library, a full-length cDNA (ZnF20) was isolated that w as 4333 bp in length and contained an open reading frame of 1029 amino acids, The ZnF20 cDNA hybridized to multiple transcripts in a thyroid cancer cell line (8.0, 4.5 and 2 kb) that increased after cycloheximi de treatment and decayed <2 h after addition of actinomycin D, The ZnF 20 mRNA was overexpressed in three of six thyroid papillary carcinomas as compared with paired normal thyroid tissue controls, The data pres ented here support the use of a targeted DRD approach for the isolatio n of rapid turnover mRNAs, many of which may be interesting candidate oncogenes.