ASSESSMENT OF TRANSCRIPT POLYADENYLATION BY 3' RACE - THE RESPONSE OFEPIDERMAL GROWTH-FACTOR MESSENGER-RIBONUCLEIC-ACID TO THYROID-HORMONEIN THE THYROID AND SUBMAXILLARY-GLANDS

The complementary DNA (cDNA) sequence of epidermal growth factor (EGF) indicates that its 3' untranslated region (3' UTR) is 745 bases long, with polyadenylation occurring at residue 4749. However, when we used reverse transcriptase-polymerase chain reaction (RT-PCR) with an anch ored 3' primer [3' rapid amplification of cDNA ends (RACE)] to amplify the 3' ends of cDNA, we actually detected two major products [800 and 600 base pairs (bp)] and a minor product (400 bp) in the thyroid or s ubmaxillary glands (SMGs) of male mice. Analysis of genomic DNA with a battery of primer pairs gave only the predicted PCR products from the 3' UTR, demonstrating the lack of introns in this region of genomic D NA and eliminating alternate splicing as the explanation of the transc ript diversity we detected. We confirmed that two potential polyadenyl ation sites proximal to residue 4749 are used in vivo by hybridizing t he same 3' RACE products with probes specific for the 5' end of the 3' UTR, and also for poly-A tails. To assess the distribution of poly-A tail lengths on transcripts using the terminal polyadenylation site (4 749), we used several different approaches to analyze 3' RACE products . Solution hybridization with 3' UTR probes revealed a striking differ ence between transcripts in SMG and thyroid: SMG contained two large 3 ' RACE populations (similar to 770 and 870 bp), whereas thyroid only c ontained one (similar to 770 bp). EGF transcript heterogeneity due to different poly-A tail lengths was confirmed using an upstream primer 4 00 bases closer to the 3' end of the 3' UTR, and TaqI digestion. Again we found two major populations in SMG (similar to 380 and 480 bp), bu t only one (380 bp) in thyroid, which upon TaqI digestion showed tissu e-specific heterogeneity only in the 3' fragment. T-4 treatment of mal e mice (0.25 mu g T-4/gm ip) increased the intensity of both populatio ns in SMG and the smaller population in thyroid within 24 h. However, after a week of T-4 injections, only the intensity of the population w ith the longer poly-A tails in the SMG remained elevated, a finding co nsistent with tissue-specific enhanced stability of transcripts due to polyadenylation. Finally, to resolve poly-A tail lengths more precise ly, we used an upstream primer that was specific for the 3' end of mur ine 3' UTR. This approach revealed that the thyroid contains three maj or populations of EGF transcripts, with poly-A tail lengths of approxi mately 20, 50, and 70 As. After T-3 treatment for 24 h, the intensity of transcripts containing 20 A's increased 52% (P < 0.02) and those wi th 50 A's increased 130% (P < 0.01), whereas there was no change in tr anscripts with tails greater than or equal to 70 As. On the other hand , there were no distinct bands in SMG samples, but rather a heterogene ous distribution of poly-A tail lengths from approximately 20-120 A's that showed an overall increase of approximately 60% in response to T- 3.