title: rRNA-complementarity in the 5' untranslated region of mRNA specifying the Gtx homeodomain protein: evidence that base- pairing to 18S rRNA affects translational efficiency.
abstract: 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.
authors.affiliation: Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Hu
authors.forename: P
authors.initials: P
authors.lastname: Tranque
authors.forename: G M
authors.initials: GM
authors.lastname: Edelman
authors.forename: V P
authors.initials: VP
authors.lastname: Mauro
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015347:RNA Probes; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016914:Ribonuclease H; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic