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

摘要

Numerous eukaryotic mRNAs contain sequences complementary to segments of the 18S and 28S rRNAs. Previous studies raised the possibility that these complementarities might allow mRNA–rRNA interactions and affect rates of translation. In the present study, we investigated the mRNA encoding the mouse Gtx homeodomain protein. This mRNA contains within its 5′ untranslated region (UTR) a segment that is complementary to two regions of the 18S rRNA, located 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 localized the cross-linked probe to the complementary segment of 18S rRNA at nucleotides 1104–1136 but not at nucleotides 701–741. To determine whether complementarity in the Gtx mRNA affected translation, a mutational analysis was performed with a Gtx-luciferase fusion construct and four related constructs with altered complementarity to the 18S rRNA. These constructs were examined for their ability to be translated in cell-free lysates prepared from P19 embryonal carcinoma and C6 glioma cell lines and after cellular transfection into these same cell lines. In both cell-free translation and transfection studies, the rate of translation decreased more than 9-fold as the degree 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.