Proteins Binding to 5' Untranslated Region Sites: a General Mechanism for Translational Regulation of Mrnas in Human and Yeast Cells RENATA STRIPECKE,' CARLA C

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1994, p. 5898-5909 Vol. 14, No. 9 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology Proteins Binding to 5' Untranslated Region Sites: a General Mechanism for Translational Regulation of mRNAs in Human and Yeast Cells RENATA STRIPECKE,' CARLA C. OLIVEIRA,2 JOHN E. G. McCARTHY 2 AND MATTHIAS W. HENTZEI* Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg,' and Department of Gene Expression, Gesellschaft fiir Biotechnologische Forschung mbH, D-38124 Braunschweig,2 Germany Received 1 April 1994/Returned for modification 20 May 1994/Accepted 31 May 1994 We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human UlA protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins. A rapidly increasing number of examples provide evidence L32 yeast ribosomal protein (10) and proteins acting on that gene expression can be regulated in the cytoplasm of Drosophila spermatogenesis (47) are possible candidates for eukaryotic cells at the level of mRNA stability, translation, or repressors functionally similar to IRP. In contrast, thymidylate the subcellular localization of mRNAs (reviewed in references synthase binds to its own mRNA 75 nucleotides downstream 11, 36, and 46). Common to these modes of posttranscriptional from the cap structure (9), a position from which IRP functions control is the role played by specific interactions between inefficiently (14, 15). LOX-BP binds to the 3' UTR of erythroid cis-acting sequences contained in the mature mRNAs and 15-lipoxygenase mRNA and represses its translation (40), regulatory RNA-binding proteins. Most biochemical informa- whereas IRP binding to an IRE in the 3' UTR fails to affect tion pertaining to cytoplasmic gene regulation by RNA-protein translation (7). Thus, it is doubtful whether the mechanism of interactions addresses translational control. Most commonly, action of thymidylate synthase and LOX-BP is similar to that the regulatory RNA sequences are found in the untranslated of IRP. Comparison of the systems that may function in a way regions at the 5' and 3' ends of the transcripts (26, 35). similar to that of IRP with those that seem to function In vertebrate cells, the regulation of ferritin and erythroid differently provides few clues to the mechanisms involved. The 5-aminolevulinate synthase mRNAs by iron has served as a paucity of well-studied physiological examples of repressor model system to elucidate how the binding of iron regulatory protein-binding site effector pairs thus led us to search for an protein (IRP; formerly known as IRF, IRE-BP, FRP, or P90) alternative approach to uncover mechanistic principles for to an RNA element (the iron-responsive element [IRE]) in the translational control. 5' untranslated region (UTR) of an mRNA controls the We asked whether RNA-binding proteins with physiological initiation step of translation. When IRP binds to an IRE, it roles unrelated to eukaryotic mRNA translation would func- represses the translation of the downstream open reading tion as translational repressor proteins when a specific mRNA frame both in vivo and in vitro (14, 18, 56). No sequences other binding site was present in a position similar to that of the IRE than the IRE are required. For an IRE-IRP complex to in ferritin mRNA. Cell-free translation experiments had sup- efficiently inhibit protein synthesis, the IRE must be localized ported the feasibility of this approach (53). We demonstrate in in a cap-proximal position of the mRNA (14, 15), whereas the this report that human and yeast cells respond to the expres- distance between the IRE and the AUG appears to be of little sion of an RNA-binding protein by strongly diminished trans- functional relevance (15, 23). lation of those mRNAs that carry a binding site for these Numerous examples of translational regulation by specific proteins in their 5' UTRs. These findings provide a basis for repressor proteins in prokaryotes exist. In most cases, the the construction of heterologous regulatory systems acting at repressor proteins directly or indirectly occlude the Shine- the translational level in eukaryotic cells. At the same time, Dalgarno sequence and/or the initiation codon (reviewed in they suggest new strategies that can be used to clone cDNAs references 13 and 34). More recently, further examples of for (regulatory) RNA-binding proteins and to study RNA- repressor proteins that, like IRP, bind to specific RNA regu- protein interactions in vivo. latory sequences have been identified in eukaryotic cells. The MATERIALS AND METHODS * Corresponding author. Mailing address: Gene Expression Pro- gramme, European Molecular Biology Laboratory, Meyerhofstrasse 1, Construction of recombinant plasmids. The plasmids used D-69117 Heidelberg, Germany. Phone: (49)-6221-387 501. Fax: (49)- to express RNA-binding proteins in yeast under control of the 6221-387 518. GAL::PGK fusion promoter are derived from YCpCATex 5898 VOL. 14, 1994 TRANSLATIONAL CONTROL BY mRNA-PROTEIN INTERACTIONS 5899 (39), which contains URA3 as a selection marker. For con- the lithium acetate procedure as previously described (21). For structing YCp-UlA, pGEM-A (2) was digested with StyI, the induction of the GAL promoter, overnight inocula were grown ends were blunt ended, and the 900-bp fragment was intro- at 30°C in lactate medium (pH 4.5) containing 0.84% (vol/vol) duced into the blunt-endedXhoI and XbaI sites of YCpCATex. sodium lactate (60%, wt/vol; Sigma), 1.13% (vol/vol) lactic For the construction of pCT1-5' containing the bacteriophage acid, 0.67% (wt/vol) yeast nitrogen base without amino acids, MS2 coat protein (CP) gene with a mammalian consensus 0.05% glucose, 0.05% yeast extract, and the corresponding sequence for efficient translation initiation (31), pCT1 (41) was amino acids or nucleotides. Induction was performed by digested with PstI and SalI and ligated to annealed and diluting the culture to an optical density at 600 nm of 0.1, phosphorylated complementary oligonucleotides containing adding 1/10 volume of 20% galactose, and continuing incuba- the sequence 5' GGATCCTCGA GCCACCAUGG CTTCTA tion at 30°C for the desired time. Protein extract preparation ACT-T TACTCAGTTC GTTCTCG 3' (underlining refers to followed the procedure described by Kingsman et al. (28) with the mammalian consensus sequence for efficient translation minor modifications. Culture (5 to 15 ml; approximate optical initiation [31]). The plasmid pCT1-5' was digested with KpnI, density at 600 nm = 0.8) was harvested by centrifugation, blunt ended, and digested with XhoI, and the 400-bp fragment washed once with 10 ml of distilled water, and suspended in 1.0 was introduced into YCpCATex by using the XhoI-XbaI sites ml of yeast lysis buffer (100 mM NaCl, 50 mM Tris-HCI [pH to generate YCp-CP. The yeast indicator plasmids are driven 7.4]). The suspension was briefly centrifuged again and resus- by the TEF1 (translation elongation factor) promoter and pended in 200,ul of lysis buffer containing 1 mM phenylmeth- contain the TRP1 selection marker. Oligonucleotides corre- ylsulfonyl fluoride, 10 ,ug of leupeptin per ml, and 1 mM sponding to the RNA-binding sequences for UlA and CP (see dithiothreitol. Cells were disrupted with glass beads at 4°C by Fig. 1) were cloned into the Aflll site present in the luciferase three cycles of vortexing for 1 min and incubation on ice for 1 leader sequence. The indicator constructs were confirmed by min. After centrifugation for 2 min at 6,000 x g, the superna- sequencing. tant was collected into a fresh tube and centrifuged for 5 min The constructs for the expression of RNA-binding proteins at 15,000 x g. The supernatant soluble extract was kept on ice in mammalian cells are derived from pSG5 (19), which con- or frozen at -80°C. Luciferase assays were performed as tains the simian virus 40 early promoter, intron 2 from the rat described by Brasier et al. (4). P-globin gene, a multiple cloning site, and the simian virus 40 Gel retardation assays. 2P-labeled RNA probes (specific polyadenylation signal. EcoRI fragments harboring the UlA activity -S x 106 cpm/,ug) containing the binding sites for (50) coding sequences were subcloned into the EcoRI site of UlA and MS2-CP were made by digesting UlA-CAT and pSG5 to generate pSG5-U1A. The MS2-CP gene containing MSC-CAT (53) with XbaI and cotranscriptionally labeling the the eukaryotic translation initiation consensus was obtained by RNA transcripts with T7 RNA polymerase. In vitro transcrip- digesting pCT1-5' with KpnI, blunt ending, and subsequently tion products were gel purified, phenol-chloroform extracted digesting with BamHI. The resulting 400-bp fragment was twice, ethanol precipitated, washed, and finally resuspended in introduced into pSG5 by using the BamHI and blunt-ended H20. Aliquots (2.5 to 5.0 ,ug each) of yeast extracts were BglII sites to yield pSG5-CP. incubated for 30 min at room temperature with 0.5 x 104 to 1 The indicator constructs used in mammalian cells are de- X 104 cpm of probe in 10 to 15 RI of the corresponding binding rived from D4-GH (also called L5-GH [7]), which contains buffer.

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