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The 3' Untranslated Region of the Human Interferon-Fl Mrna Has an Inhibitory Effect on Translation (SP6 RNA Transcripts/Translational Efficiency) V

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The 3' Untranslated Region of the Human Interferon-Fl Mrna Has an Inhibitory Effect on Translation (SP6 RNA Transcripts/Translational Efficiency) V Proc. Natl. Acad. Sci. USA Vol. 84, pp. 6030-6034, September 1987 Biochemistry The 3' untranslated region of the human interferon-fl mRNA has an inhibitory effect on translation (SP6 RNA transcripts/translational efficiency) V. KRUYS*, M. WATHELET*, P. POUPARTt, R. CONTRERASt, W. FIERSt, J. CONTENTt, AND G. HUEZ*§ *D6partement de Biologie Moldculaire, Universitd Libre de Bruxelles, 1640 Rhode-St-Gen~se, Belgium; tf~partement de Virologie, Institut Pasteur du Brabant, 1180 Bruxelles, Belgium; and fLaboratory of Molecular Biology, State University of Ghent, 9000 Ghent, Belgium Communicated by M. Van Montagu, April 13, 1987 (received for review January 20, 1987) ABSTRACT In vitro-transcribed human interferon-I3 transfection of the IFN-,8 gene, that the 3' region of the (IFN-1P) mRNA, which contains all the sequence of the natural mRNA influences its stability. Nevertheless, the authors molecule, is poorly translated in a reticulocyte lysate or when could not conclude whether the coding sgequence or the 3' injected in Xenopus oocytes. This low level of translation is due UTR is responsible for the destabilization of the mRNA. to an inhibition by the 5' ;and 3' untranslated regions (UTRs). It has been shown that the IFN-,8 mRNA is posttranscrip- Indeed, the replacement of these sequences by those ofXenopus tionally regulated (7). During the course of a study on the .-globin mRNA dramatically increases the translational effi- possible mechanism of such regulation, we observed that an ciency of the mRNA, especially in oocytes. This phenomenon is in vitro-transcribed IFN-p3 mRNA was actually very poorly not due to a difference in mRNA stability since both native and translated in certain translation systems and that this phe- chimeric mRNAs remain undegraded, at least during 'the nomenon was not due to a rapid degradation of the molecule. translation period considered. Construction of different chi- We demonstrate here that the IFN-P UTRs, and especially meric molecules having various combinations of5' and 3' UTRs the 3' region, are inhibitory for the m1NA translation. Such from IFN-J3 or Xenopus ,.1-globin mRNA or a small sequence of inhibition occurs even when this 3' sequence is added to a SP6 polylinker as 5' UTR has revealed that the 3' UTR ofIFN-f3 completely different mRNA, that of chicken lysozyme. in itself has a pronounced inhibitory effect on translation in the two translation systems from animal cells. Indeed, the addition MATERIALS AND METHODS of this 3' UTR at the 3' end of the coding region of a chicken lysozyme mRNA also causes a large decrease of its translational Construction of the Different Plasmids Used for in Vitro capacity in both systems. However, the nature of-the 5' Synthesis of Corresponding RNAs. Hu-IFN-f3 mRNA is ob- noncoding sequence influences the degree of translation inhi- tained by in vitro transcription of a Sal I linearized SP65 bition exerted by the 3' UTR. Remarkably, we observed no vector (SP65IFN/3) containing the Hae III/HindII fragment difference in translation level when the different mRNAs were of the Hu-IFN-f3 gene inserted downstream of the SP6 tested in a wheat germ extract. promoter. This Hae III/HindIl fragment corresponds to the complete Hu-IFN-p8 cDNA sequence (8). The IFN-,B in Eukaryotic messenger mRNAs contain both 5' and 3' un- vitro-transcribed mRNA from this SP65IFN8 plasmid con- translated regions (UTRs) of different sizes. Most mRNAs tains in its 5' UTR, in addition to the natural 5' IFN-,8 mRNA also possess a poly(A) segment at their 3' end. There is some UTR, 19 bases corresponding to a gene segment upstream of evidence that those'untranslated sequences are involved in the cap site and 21 nucleotides due to the transcription of the posttranscriptional controls of gene expression either by vector polylinker. governing mRNA translation efficiency or stability. For IFN-,f mRNA with its 5' and 3' UTRs replaced by those of example, the poly(A) segment markedly increases the sta- P-globin mRNA is synthesized by transcription of a plasmid bility of several mRNAs introduced either in oocytes or derived from that of Krieg and Melton (SP64TIFN,8) (9). somatic cells in culture (1). On the other hand, the real Cytidine residues present at the 3' end of the SP64TIFN8 meaning of the presence of rather long 5' and 3' UTRs were eliminated as follows: the SP64TIFNB was linearized by adjacent to the coding part is still unclear. It has been shown HindIII, which cleaves 7 base pairs (bp) upstream of the 5' that some sequences of limited length in the 5' UTR play an UTR. The extremities were then filled in with the Klenow important role in translation. The "Kozak" sequence and the was Sal which CCRCCATGG (where R = purine) appears to be required for polymerase plasmid digested by I, the most efficient recognition of the correct initiation codon cleaves it 3 nucleotides downstream of the 3' UTR. The by the ribosomes in eukaryotic cells (2). The length of the 5' resulting 850-bp fragment corresponding to the complete untranslated sequence in itself does not influence the trans- IFN-p coding sequence flanked by the P-globin UTRs was lation capacity, at least for some mRNAs (3). However, the inserted in the SP65 vector between the EcoRI filled-in and translation of the mRNA can be markedly impaired if a very Sal I sites. RNA was transcribed from this construction stable hairpin structure is introduced in the 5' UTR (4). Data (SP65IFNB 5'-3'-GlobpC) previously linearized by Sal I. concerning the 3' UTR role remain somewhat contradictory. This RNA was then transcribed into cDNA using oligo(dT) as For example, Soreq et al. (5) have reported that the removal primer. The cDNA was digested by Pst I, which cleaves in of most of the 3' UTR from the mRNA' coding for human the coding sequence (200 bp from the AUG). It was then interferon-,B (Hu-IFN-,3) does not affect either the stability or inserted in SP65IFNP 5'-3'-GlobpC. For this, the latter the translation of the mRNA in Xenopus oocytes. On the plasmid was first linearized with HindIII then filled in and other hand, using Vero cells, which present an IFN-,B redigested with Pst I. The resulting construction (SP65IFNp production deficiency, Mosca et al. (6) demonstrated by 5'-3'-G) was linearized by HindIII for in vitro transcription. The publication costs of this article were defrayed in part by page charge Abbreviations: IFN-,B, interferon-/3; Hu-IFN, human IFN; UTR, payment. This article must therefore be hereby marked "advertisement" untranslated region. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6030 Downloaded by guest on September 26, 2021 Biochemistry: Kruys et al. Proc. Natl. Acad. Sci. USA 84 (1987) 6031 The mRNA with a 19-base synthetic oligonucleotide as 5' UTR was synthesized by transcription of a plasmid contain- Ad...-i ing the HindII/HindII fragment of the IFN-,f gene (SP65IFN,8 5'-SO). This fragment corresponds to the coding F 'a ._. and 3' noncoding sequence of the IFN-83 mRNA. The construction was obtained by insertion of this HindII/HindII _ A: with EcoRI linkers into the SP65 ,,K,,_s ,. .. ' ' ...:... fragment EcoRI-digested vector. Before transcription, the SP65IFNf3 5'-SO was lin- ;...Xv,.,'v1>g. s earized by Sac I, which cleaves 7 bp downstream of the 3' 1 2 3 4 ...> :- :.....e. 7 8 noncoding sequence. SP65IFN,8 pla'smid containing the 3' ,-globin UTR (SP65IFN,8 3'-G) was constructed as follows. FIG. 1. Translation ofthe two in vitro-transcribed IFN-,8 mRNAs For technical convenience-e.g., the presence of suitable that have the most different translational efficiency: SP65IFN,8 and restriction sites-all constructions containing the 3' 3-globin SP65IFN,8 5'-3'-G in the three different systems. Wheat germ were from the SP64TIFNJ3 extract: lane 1, SP65IFN,8; lane 2, SP65IFNJ 5'-3'-G. Reticulocyte UTR synthesized starting plas- lysate: lane 3, SP65IFNf 5'-3'-G; lane 4, SP65IFNp. Oocytes: lane mid, which has an extra oligo(C). We checked that the 5, SP65IFN,8 5'-3'-G; lane 6, SP65IFN,8 (the three bands correspond removal ofthis oligo(C) sequence ohly marginally influences, to different stages ofglycosylation). Oocyte incubation medium: lane if at all, the translational efficiency of the mRNA (data not 7, SP65IFN,8 5'-3'-G; lane 8, SP65IFN/3. shown). SP64TIFN,8 was digested by Pst I to isolate the 610-bp insert corresponding to part of the IFN-,8 coding sequence as well as the ,-globin 3' UTR. This insert was (14). Analysis of the translation products was performed on ligated to the SP65IFN,8 plasmid previously digested by Pst NaDodSO4/20% polyacrylamide gels. I to remove the 600-bp fragment corresponding to the same Injection into Oocytes. Oocytes were injected with 50 n1 of coding region as above but followed by the IFN-p8 3' UTR. mRNA dissolved in water and adjusted to a concentration of The other two constructions-SP64IFN,3 5'-G and SP65- 0.1 mg/ml. The injection procedure and the incubation ofthe IFN/3 5'-SO 3'G-were obtained using the same strategy oocytes have been described by Gurdon et al. (15). After starting from SP65IFN/3 and SP65IFN,8 5'-SO constructions. injection, the oocytes were incubated for 6 hr at 18TC in These three plasmids were linearized, respectively, by Barth's medium (0.01 ml per oocyte) containing [35S]methi- HindIII, Sal I, and HindIII, which cleave at the nearest sites onine (9 uCi per oocyte), 10% bovine serum albumin, 1% downstream of the insert.
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