Fig. 2. Interaction of POS-1 and SPN-4 with glp-1 3' UTR. (A) The glp-1 mRNA 3' UTR. The entire 3' UTR is 369 bases long from the stop codon UAA to the poly A addition site. The SCR was originally defined as lying between bases 180 and 240, and the TCR between bases 245 and 369 (Evans et al., 1994). The comparison of the 3' UTRs of closely related species (Rudel and Kimble, 2001) suggested some minor modifications on the regions as follows: modified SCR was between bases 172 and 237, and modified TCR was from base 242. Thus, the regions are as follows: the 294-base (bases 26 to 319), the 138-base (bases 26 to 163, SCR (172 to 237) and the TCR*(bases 242 to 319). TCR* indicates a subregion of TCR. (B) Outline of the structure of POS-1. Two CCCH zinc fingers are located in the middle of the 264 amino acid POS-1 protein. The allele ne51 is a missense mutation at codon 147, C(TGC) to Y(TAC), in the second zinc finger. (C) Yeast tri-hybrid analysis. Based on the reporter RNA study (Evans et al., 1994) and the comparative study (Rudel and Kimble, 2001), the following regions were taken from the 369-base glp-1 mRNA 3' UTR for the construction of hybrid RNA with RRE, a Rev responsive element recognized by HIV-1 RevM10 (Putz et al., 2000): row 1, bases 26-319 for 138-base SCR-TCR* (the entire region); row 2, bases 26-163 for 138-base (non SCR, non TCR region); row 3, bases 172-237 for the SCR; and row 4, bases 242-319 for the TCR* (TCR subregion). Row 5 is a negative control RNA. C indicates growth on a non-selection medium without 3AT; S indicates growth on a selection medium containing 50 mM (for POS-1) or 10 mM (for SPN-4) 3-AT. (D) Western blot analysis of the tri-hybrid strains by using an anti-POS-1 antibody: (1) GAL4 AD, (2) GAL4 AD::POS-1 and (3) GAL4 AD::POS-1(C147Y) were expressed in yeast. In all the yeast strains, RRE-138-base SCR-TCR* was co-expressed. POS-1(C147Y) was expressed at the same level as POS-1.