rRNA-complementarity in the 5 ' untranslated region of mRNA specifying theGtx homeodomain protein: Evidence that base-pairing to 18S rRNA affects translational efficiency

Numerous eukaryotic mRNAs contain sequences complementary to segments of th e 18S and 28S rRNAs. Previous studies raised the possibility that these com plementarities might allow mRNA-rRNA interactions and affect rates of trans lation. In the present study, we investigated the mRNA encoding the mouse G tx homeodomain protein. This mRNA contains within its 5' untranslated regio n (UTR) a segment that is complementary to two regions of the 18S rRNA, loc ated at nucleotides 701-741 and 1104-1136. A Gtx RNA probe containing this complementarity could be photochemically cross-linked to ribosomal subunits through a linkage to 18S rRNA but not to 28S rRNA Oligonucleotide-directed RNase H digestion of the rRNA and a reverse transcription analysis localiz ed the cross-linked probe to the complementary segment of 18S rRNA at nucle otides 1104-1136 but not at nucleotides 701-741. To determine whether compl ementarity in the Gtx mRNA affected translation, a mutational analysis was performed with a Gtx-luciferase fusion construct and four related construct s with altered complementarity to the 18S rRNA. These constructs were exami ned for their ability to be translated in cell-free lysates prepared from P 19 embryonal carcinoma and C6 glioma cell lines and after cellular transfec tion into these same cell lines. In both cell-free translation and transfec tion studies, the rate of translation decreased more than 9-fold as the deg ree of complementarity to nucleotides 1104-1136 of the 18S rRNA increased. We hypothesize that segments complementary to rRNA, such as those contained within the Gtx mRNA, form a category of cis-acting regulatory elements in mRNAs that affect translation by base pairing to rRNA within ribosomes.