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Functional Mapping of Destabilizing Elements in the Protein-coding Region of the Drosophila fushi tarazumRNA

2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês

10.1074/jbc.m102965200

1083-351X

Jun-itsu Ito, Marcelo Jacobs‐Lorena,

Genomics and Chromatin Dynamics

The instability of the fushi tarazu(ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3′ untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3′ UTR of the hybrid mRNAs. We conclude that ftzmRNA has three redundant instability elements, two in the protein-coding region and one in the 3′ UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5′-elements depended on their position within the message. The instability of the fushi tarazu(ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3′ untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3′ UTR of the hybrid mRNAs. We conclude that ftzmRNA has three redundant instability elements, two in the protein-coding region and one in the 3′ UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5′-elements depended on their position within the message. untranslated region polymerase chain reaction kilobase(s) aldolase Drosophila embryonic development depends on the precise temporal and spatial expression of maternal and zygotic pattern-forming genes (1Ingham P.W. Nature. 1988; 335: 25-34Crossref PubMed Scopus (616) Google Scholar). Maternal pattern-forming genes are transcribed during oogenesis, and their mRNA abundance decreases rapidly in the early embryo. Moreover, many mRNAs encoded by zygotic pattern-forming genes undergo dramatic changes in abundance and spatial distribution during early embryogenesis. To achieve these rapid changes, especially for rapid down-regulation, transcriptional control alone is insufficient, and regulation at the level of mRNA stability is essential. For instance, the maternal bicoid mRNA is completely stable during the first 2 h of embryogenesis but is rapidly destabilized at cellularization of the blastoderm (2Surdej P. Jacobs-Lorena M. Mol. Cell. Biol. 1998; 18: 2892-2900Crossref PubMed Google Scholar). As discussed in the following text, the zygotic fushi tarazu(ftz) mRNA is one of the most unstable eukaryotic mRNAs known. Given that most mRNAs in the Drosophilaembryo are constitutively stable (30Fontes A.M. Ito J. Jacobs-Lorena M. Curr. Top. Dev. Biol. 1999; 44: 171-202Crossref PubMed Scopus (14) Google Scholar), the question arises how selected mRNAs in the same embryo cytoplasm are targeted for degradation. Recognition of the targeted RNAs by the RNA degrading machinery must involve cis-acting sequences. These sequences are the focus of the present study. ftz is a member of the pair-rule class of segmentation genes and one of the best characterized early zygotic genes. In early embryosftz mRNA is detected only from about 1.5 to 4.5 h after fertilization. When first expressed, ftz mRNA is uniformly distributed through the embryo (4Weiner M.P. Kornberg T. Nature. 1985; 318: 433-439Crossref PubMed Scopus (78) Google Scholar). As development progresses, its distribution first becomes restricted to a region comprising from 15 to 65% of egg length, then to four broad bands, and finally to seven narrow stripes that encircle the embryo (4Weiner M.P. Kornberg T. Nature. 1985; 318: 433-439Crossref PubMed Scopus (78) Google Scholar, 5Hafen E. Kuroiwa A. Gehring W.J. Cell. 1984; 37: 833-841Abstract Full Text PDF PubMed Scopus (204) Google Scholar, 6Yu Y. Pick L. Mech. Dev. 1995; 50: 163-175Crossref PubMed Scopus (45) Google Scholar). The seven stripes are short-lived, and no ftz mRNA is detected by 5 h after fertilization. This rapid change of expression pattern and formation of stripes in a short time span can be attributed to the termination of transcription in interstripe regions coupled with rapid mRNA turnover. The need for rapid mRNA turnover is emphasized by the fact that the FTZ protein activates its own transcription in a positive feedback loop (7Hiromi Y. Gehring W.J. Cell. 1987; 50: 963-974Abstract Full Text PDF PubMed Scopus (247) Google Scholar). Thus, it is important during the evolution of the spatial pattern of ftzexpression that both mRNA and protein be rapidly cleared from the interstripe regions. Edgar et al. (8/deleted in proof.Google Scholar) measured ftz mRNA stability in embryos and found that its half-life changes from 14 min at 2.5 h to 6 min at 4 h after fertilization. This makesftz mRNA one of the shortest-lived mRNAs among higher eukaryotes (3Deleted in proof.Google Scholar). Stabilization of ftz mRNA and FTZ protein results in developmental delay and defects, suggesting thatftz mRNA and FTZ protein instability are crucial for normal development (9Edgar B.A. Odell G.M. Schubiger G. Genes Dev. 1987; 1: 1226-1237Crossref PubMed Scopus (63) Google Scholar, 10Kellerman K.A. Mattson D.M. Duncan I. Genes Dev. 1990; 4: 1925-1935Crossref PubMed Scopus (63) Google Scholar, 11Welte M.A. Duccan I. Lindquist S. Genes Dev. 1995; 9: 2240-2250Crossref PubMed Scopus (24) Google Scholar). Earlier studies from this laboratory using hybrid genes that fuseftz sequences to the stable ribosomal protein A1(rpA1) mRNA provided evidence for at least two destabilizing elements in ftz mRNA (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). One consisted of a 201-nucleotide element, including an essential 68-nucleotide sequence, located in the 3′ UTR1 and termed FIE3 (f tz instabilityelement 3′). The other element(s) were assigned to the 5′-one-third of the ftz mRNA but remained otherwise uncharacterized. Here we report on the identification of two separate 5′-instability elements within the first 600 nucleotides of theftz mRNA. Both 5′-elements are located within the protein-coding region of the message. Each 5′-instability element is sufficient to destabilize the stable rpA1 mRNA, but the destabilizing activity is dependent on their position within the mRNA. The first letter of the name of each construct designates the promoter that drives it. Thus, F = ftz, r =rpA1, and S = sgs-3 promoter attached to the hsp26 nurse cell enhancer. Numbering in the following text uses +1 as the position of transcription initiation, which corresponds to position 901 in the sequence deposited in GenBankTM (accession numbers X00854and K01951). We found that ftz genomic clone λA439 (13Weiner A.J. Scott M.P. Kaufman T.C. Cell. 1984; 37: 843-851Abstract Full Text PDF PubMed Scopus (50) Google Scholar) had a nine-nucleotide deletion in the protein-coding region (from 260 to 268) when compared with the sequence deposited in the data base. To resolve this discrepancy, the ftz 5′-region was amplified by PCR using DNA from yellow-white flies, the recipient strain for P-element transformation. Sequencing revealed that the same nine nucleotides were missing. Moreover, with the exception of the nine-base deletion and four one-base polymorphisms (C instead of T at 126 and 222, C instead of A at 516, and G instead of C at 551), the sequence of the entire 5′-one-third of the ftz mRNA (from 1 to 636) matched the sequence deposited in GenBankTM. These hybrid genes were reported previously (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). Briefly, the 7.9-kb KpnI-SalI fragment (Ff5) and the 4.0-kbSalI-KpnI fragment (f3) were obtained from theftz genomic clone λA439 (13Weiner A.J. Scott M.P. Kaufman T.C. Cell. 1984; 37: 843-851Abstract Full Text PDF PubMed Scopus (50) Google Scholar). The 1.1-kbBamHI-SalI fragment (Rr5) and the 1.3-kbSalI-BamHI fragment (r3) were obtained from the plasmid pD5 (14Qian S. Zhang J.-Y. Kay M.A. Jacobs-Lorena M. Nucleic Acids Res. 1987; 15: 987-1003Crossref PubMed Scopus (56) Google Scholar) that contained the 2.4-kb BamHI fragment of the rpA1 gene. Each fragment of ftz and therpA1 gene was combined reciprocally and subcloned into pGEM3 (Promega). The 0.7-kb fragment (from 856 to 1557, where +1 is the transcription initiation site) of rat aldolase B(aldB) cDNA (15Tsutsumi K. Mukai T. Tsutsumi R. Mori M. Daimon M. Tanaka T. Yatsuki H. Hori K. Ishikawa K. J. Biol. Chem. 1984; 259: 14572-14575Abstract Full Text PDF PubMed Google Scholar) was synthesized by PCR with primers ald1 and ald2 (Table I). This PCR fragment contained the 3′-one-third of the protein-coding region and entire 3′ UTR of the aldB cDNA, including the polyadenylation signal (16Tsutsumi K. Mukai T. Tsutsumi R. Hidaka S. Arai Y. Hori K. Ishikawa K. J. Mol. Biol. 1985; 181: 153-160Crossref PubMed Scopus (53) Google Scholar), provided by the primer ald2. The PCR fragment was cloned into the pGEM-T Easy vector (Promega), digested with EcoRI, and inserted into the EcoRI site of the 3′ UTR of f3. In turn, this f3+aldB fragment was fused to Ff5 to produce Fftz+aldB. A f3-FIE3 fragment that lacks the 201-base pair FIE3 sequence was obtained as previously described (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). Fftz+aldB-FIE3 was constructed as described for Fftz+aldB, except that f3-FIE3 was used instead of f3.Table IOligonucleotides used for making constructsOligonucleotideSequence1-aBoldface type indicates endonuclease restriction sites. Underlining indicates added translation initiation sequences.Restriction enzymeGene (positions1-bPositions in parenthesis reveal the position of the mRNA; the transcription initiation site is +1.)Primer 15′-TTGCCGGCAGGGCTCTCTGATTTTGCTA-3′NgoMIftz (1–20)Primer 25′-TTGCCGCCTCGACATCCTCGGAGGCGC-3′NgoMIftz (636–616)Primer 35′-TTGCCGGCTACTACGATAATTCAGGCAGC-3′NgoMIftz (211–231)Primer 45′-TTGCCGCCGATGGAAGCAGCATCATATTC-3′NgoMIftz (423–403)Primer 55′-TTGCGGCCGCAGGGCTCTCTGATTTTGCTA-3′NotIftz (1–20)Primer 65′-CTTGATGACCTTGGTCAGACG-3′rpA1 (221–201)Primer 75′-ATGCGGCCGC GATATGGATAATTCAGGCAGCAATGC-3′NotIftz (217–236)Primer 85′-CTGTCGACGATGGAAGCAGCATCATCTTC-3′SalIftz (423–403)Primer 95′-ATGCGGCCGC GATATGGATGATGCTGCTTCCATCAT-3′NotIftz (406–425)Primer 105′-GTGTCGACGTTCTGATAGTAGGCATTGC-3′SalIftz (249–230)Primer 115′-AAGCGGCCGC GATATGGAGAGCTGCTACTACTACAAC-3′NotIftz (301–321)Primer 125′-TTGTCGACTTGCACGGGCGGTACAGTCTG-3′SalIftz (363–343)Primer 135′-TTGCGGCCGC GATATGGTCGAGCAGGTGAAGAAGGCT-3′NotIftz (520–540)Primer 145′-TTGTCGACGTCGTAGCTGGGAGCGGG-3′SalIftz (588–571)Primer 155′-TTGCGGCCGC GATATGGCCTACTATCAGAACACCTC-3′NotIftz (235–254)Primer 165′-TTGTCGACGCTCTCCGAGTAACTCTCCTG-3′SalIftz (306–286)Primer 175′-TAGCGGCCGC GATATGGCTCCCAGCTACGACCAAGA-3′NotIftz (574–593)Primer 185′-TTGTCGACCTGCTCGACGGTGGTGTAGA-3′SalIftz (528–519)Primer ald15′-TAGCCGGCCTGTGCCTAGTATCTGCTT-3′AldB (858–884)Primer ald25′-AGTACCAACTGAAGGTTCATAGACTTTTTA-3′AldB (1557–1527)1-a Boldface type indicates endonuclease restriction sites. Underlining indicates added translation initiation sequences.1-b Positions in parenthesis reveal the position of the mRNA; the transcription initiation site is +1. Open table in a new tab The PCR products synthesized from Ff5r3 with primers 1 and 2, 1 and 4, and 3 and 2 (Table I) were digested with NgoMI and inserted into the NgoMI site in the 3′ UTR of the rpA1 gene in plasmid pD5. They were named Rr-abc, Rr-ab, and Rr-bc, respectively. PCR products were synthesized with the following primers (Table I): Ss-abc, primers 5 and 6; Ss-ab, primers 5 and 8; Ss-bc, primers 7 and 6; Ss-a, primers 5 and 10; Ss-b, primers 7 and 8; Ss-c, primers 9 and 6; Ss-b1, primers 7 and 12; Ss-b2, primers 11 and 8; Ss-b3, primers 15 and 16; Ss-c1, primers 9 and 14; Ss-c2, primers 13 and 6; Ss-c3, primers 9 and 18; Ss-c4, primers 13 and 14; Ss-c5, primers 17 and 6. For PCR fragments that lacked a translation initiation site, s-bc, b, c, b1–3, and c1–5, a seven-base sequence (GATATGG) was provided by the primer to obtain the same efficient translation initiation site as intact ftz mRNA (17Cavener D.R. Nucleic Acids Res. 1987; 15: 1353-1361Crossref PubMed Scopus (739) Google Scholar, 18Cavener D.R. Ray S.C. Nucleic Acids Res. 1991; 19: 3185-3192Crossref PubMed Scopus (526) Google Scholar). The PCR products were fused to r3. All constructs were sequenced to confirm that the PCR fragments had no errors and that the reading frame was maintained. Constructs Rr-abc, ab, bc, Fftz+aldB, and Fftz+aldB-FIE3 were introduced into CaSpeR vector (19Pirrotta V. Rodriguez R. Denhart D. Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Butterworth, Boston1988: 437-456Crossref Google Scholar). To provide Ss constructs with a promoter, they were introduced into the CaSpeR4/GERM4 vector (2Surdej P. Jacobs-Lorena M. Mol. Cell. Biol. 1998; 18: 2892-2900Crossref PubMed Google Scholar), which contained CaSpeR, polylinker from Gehring's pW8, a nurse cell-specific enhancer from the heat shock 26 gene hsp26, and the sgs3 promoter. All constructs (500 μg/ml) were mixed with the phs-π helper (20Steller H. Pirrotta V. Mol. Cell. Biol. 1986; 6: 1640-1649Crossref PubMed Scopus (77) Google Scholar) (100 μg/ml) and injected into yellow-whitemutant embryos (19Pirrotta V. Rodriguez R. Denhart D. Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Butterworth, Boston1988: 437-456Crossref Google Scholar, 21Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2318) Google Scholar). Embryos were collected on fresh yeast plates at 25 °C for 1 h and left at 25 °C to age for different lengths of time. Embryos were collected, washed with embryo washing buffer (0.5% Triton X-100, 300 mm NaCl, 10 mm Tris, pH 7.5), frozen in liquid nitrogen, and kept at −80 °C. Embryo developmental synchrony was checked by staging an aliquot of each collection at 2–3 h of development. Collections containing more than 10% older embryos (retained) or unfertilized eggs were discarded. RNA Extraction and Northern Blot Hybridization—Total RNA was isolated from frozen embryos by homogenization in TRI REAGENT (Molecular Research Center, Inc.) following the manufacturer's recommendations. Northern blot analysis was performed as previously described (22Ito J. Kuzumaki T. Otsu K. Iuchi Y. Ishikawa K. Arch. Biochem. Biophys. 1998; 350: 291-297Crossref PubMed Scopus (18) Google Scholar). Briefly, 10 μg of total RNA was fractionated by electrophoresis in 1–2% agarose gels containing 18% formaldehyde, and RNA was transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech) (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The RNAs were fixed to the membrane by UV cross-linking and hybridized with α-32P-labeled probes synthesized by random primer labeling. For hybridization with a different probe, the membrane was boiled 5 min to remove the first probe, following the manufacturer's recommendations. The plasmid pD5, containing therpA1 gene, Ff5, and f3 were used to prepare probes for Northern blot hybridization. Estimates of the half-life of mRNAs were obtained from the quantification of the radioactive signal with the Molecular Imager system (Bio-Rad). For each transgenic strain, Northern blot hybridization was repeated at least three times with RNA from separate embryo collections. The stability of transgenic mRNAs in developing embryos was measured by a strategy previously developed for this purpose (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). The critical feature is the use of promoters that are strongly expressed during oogenesis but silent in early embryogenesis. The two promoters used in this study are rpA1 (14Qian S. Zhang J.-Y. Kay M.A. Jacobs-Lorena M. Nucleic Acids Res. 1987; 15: 987-1003Crossref PubMed Scopus (56) Google Scholar) andhsp26/sgs3 (24Garfinkel M.D. Pruitt R.E. Meyerowitz E.M. J. Mol. Biol. 1983; 168: 765-789Crossref PubMed Scopus (89) Google Scholar, 25Serano T.L. Cheung H.K. Frank L.H. Cohen R.S. Gene. 1994; 138: 181-186Crossref PubMed Scopus (25) Google Scholar). The latter promoter contains the nurse cell-specific enhancer from the hsp26 gene linked to the basal sgs3 promoter. Another key feature is that theftz mRNA is stable in ovaries and is destabilized at fertilization. 2Fontes, A. M., Riedl, A., and Jacobs-Lorena, M. (2001) Genesis, in press. As a consequence, transcripts containing ftz sequences accumulate to high abundance during oogenesis and start decaying when the egg is fertilized.2 Because the rpA1 andhsp26/sgs3 promoters are silent in early embryos, decay of transgenic mRNA abundance (as measured by Northern blot analysis) serves as a direct measure of mRNA stability. This strategy avoids the use of drugs that may cause artifacts (27Shyu A.-B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (445) Google Scholar). TherpA1 mRNA is stable in both ovaries and embryos and served as an internal loading control. In an earlier study (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar), the stability of the 5′-one-third (here called f5; nucleotides 1–636) and the 3′-two-thirds (here called f3) of the ftz mRNA was investigated separately. One of these fragments (f5) was fused to the 3′-two-thirds (r3) and the other (f3) to the 5′-one-third (r5) of the stablerpA1 mRNA to yield the Ff5r3 and Rr5f3 hybrid mRNAs, respectively (Fig. 1 A). Both hybrid transcripts were unstable (Fig.2 A). Rr5f3 is transcribed maternally, and its mRNA decayed rapidly after fertilization. Ff5r3 is transcribed only transiently from the ftz promoter during early embryogenesis, and its rapid decay after 4 h of development (compare 3–4 h and 4–5 h in Fig. 2 A) indicates that this mRNA is highly unstable. We concluded that in addition to the previously characterized 201-nucleotide FIE3 in the 3′ UTR, the 5′-one-third of the ftz mRNA also contains destabilizing sequences.Figure 2Evidence for destabilizing elements in the 5′-region of the ftz mRNA. Each construct was transformed into the Drosophila germ line. Total RNAs from synchronized embryos were analyzed by Northern blot hybridization withftz and rpA1 probes. The structure of each construct is illustrated next to its name. Embryo age ranges (in h) are indicated below the autoradiograms. The rpA1 mRNA served as a loading control. A, hybridftz/rpA1 mRNAs. The r5f3 construct is driven by the rpA1 promoter, and the f5r3 construct is driven by the ftz promoter. mRNA sizes are as follows:ftz, 1.8 kb; r5f3, 1.4 kb; f5r3, 0.6 kb; rpA1, 0.6 kb.B, deletion of the FIE3 sequences from the ftzmRNA. A 700-nucleotide rat aldolase B fragment was inserted in theftz 3′ UTR to distinguish the transgenic mRNA from the endogenous ftz mRNA. The second construct differs from the first by the absence of the 201-nucleotide FIE3 element. Both constructs are driven by the ftz promoter. mRNA sizes: ftz+aldB, 2.5 kb; and ftz+aldB-FIE3, 2.3 kb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Fftz+ald-FIE3 construct provided further evidence for the presence of RNA-destabilizing sequences in f5. This construct consisted of a deletion of FIE3 from the intact ftz mRNA (Fig.1 B). This construct also contained a rat aldolase B mRNA (aldB) tag, to allow the transgenic mRNA to be distinguished on Northern blots from the endogenousftz transcript. To verify that insertion of the aldolase tag does not affect ftz mRNA stability, a control construct (Fftz+ald; Fig. 1 B) containing the intact ftzmRNA tagged with the aldB fragment was analyzed in parallel. As shown in Fig. 2 B, both constructs were unstable inDrosophila embryos, indicating that ftz mRNA contains instability elements other than FIE3. The destabilizing element of f3 is FIE3 (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). The identification and characterization of the f5-destabilizing sequences (termed FIE5) was the object of this study. Previously, we have inserted FIE3 into the 3′ UTR of the stable rpA1 gene to demonstrate that this element is sufficient for mRNA destabilization (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). We created similar constructs to investigate whether FIE5 is also sufficient for RNA destabilization. The entire f5 sequence (1), the 5′-two-thirds (1), and the 3′-two-thirds (211) were inserted into the 3′ UTR of rpA1 to yield constructs Rr-abc, Rr-ab, and Rr-bc, respectively (Fig. 1 C). Transcription in all constructs was driven by the rpA1 promoter. Surprisingly, the resulting three transcripts were stable in transgenic embryos (Fig. 3). These results suggest that FIE5 destabilizing activity is position-dependent and that unlike FIE3, FIE5 is not functional when located in the 3′ UTR. Next, we initiated a deletion analysis by placing the f5 fragment at its original position 5′ of the rpA1 sequences. Construct Ss-abc contained the whole f5 sequence (from 1 to 636), Ss-ab contained the 5′-two-thirds (from 1 to 423), and Ss-bc contained the 3′-two-thirds (from 217 to 636) (Fig. 1 D). All transcripts decayed rapidly during early embryogenesis (Fig. 4). These results suggest that FIE5 destabilizing sequences are likely to be present in the Ss-ab/Ss-bc overlap region. Alternatively, more than one destabilizing sequence may occur within f5. The results also indicate that the maternal hsp26/sgs3 promoter can be used for measurement of mRNA stability despite the presence of 41 additional sgs3-encoded nucleotides at the 5′-end of all transcripts driven by this promoter. Separate experiments showed that these 41 nucleotides contain no destabilizing sequences (cf. constructs Ss-a, Ss-b2, Ss-c3, and Ss-c5). A second generation of constructs placed each third of f5 next to 5′-rpA1 sequences (Ss-a, Ss-b, and Ss-c; Fig. 1 D). As shown in Fig.5, s-a mRNA was stable, whereas s-b and s-c mRNAs decayed rapidly during early embryogenesis. These results suggest that f5 has at least two destabilizing elements, one (FIE5-1) within fragment b (from 217 to 423) and the other (FIE5-2) within fragment c (from 406 to 636). Each element is sufficient for destabilization of an otherwise stable mRNA, and both elements are located in the protein-coding region of ftz mRNA. Additional deletion constructs (Ss-b1 to Ss-b3; Fig. 1 D) were generated to further map FIE5-1. Initial analysis of embryos carrying two overlapping constructs, Ss-b1 and Ss-b2, indicated that the s-b1 mRNA was unstable whereas s-b2 mRNA was stable (Fig.6). These results suggested that FIE5-1 is located in a region of Ss-b1 (250) that does not overlap with the two stable sequences, SS-a and SS-b2 (Fig. 1 D). This assumption was confirmed with a third construct, Ss-b3 (235), which encodes an unstable mRNA (Fig. 6). Thus, FIE5-1 is located within a 63-nucleotide sequence of the ftz protein-coding region (the stated length takes into account that our construct andyellow-white flies have nine fewer nucleotides than the sequence deposited in the data base; cf. “Experimental Procedures”). Additional deletion constructs (Ss-c1 to Ss-c5; Fig. 1 D) were also generated to further map FIE5-2. As shown in Fig. 7, the s-c1 and s-c2 mRNAs were both unstable in early embryos, indicating that an instability element is located in the region of overlap or that multiple instability elements are located in ftz fragment c. To clarify these issues, three more constructs (Ss-c3 to Ss-c5; Fig.1 D) were analyzed. Of the three constructs, only Ss-c4 (from 520 to 588) encoded an unstable mRNA (Fig. 7), indicating that FIE5-2 must reside within this 69-nucleotide region. In some experiments, the s-c3 mRNA was observed to decline slightly from 0–1 to 1–2 h but remained stable during the remaining time points (data not shown). In an earlier study (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar), we developed a new method for thein vivo analysis of mRNA stability in earlyDrosophila embryos that does not require the use of drugs or any experimental interference. This method led to the identification of a mRNA-destabilizing element (FIE3) in the ftz 3′ UTR. In this work the method was used to identify two additional instability elements, both in the 5′-protein-coding region. Hence, ftzmRNA has three redundant destabilizing elements, each of which is sufficient to promote mRNA degradation in early embryos. Although the significance of the occurrence of three redundant elements in the same message can only be speculated on, redundancy may be tied to the fact that ftz mRNA instability is crucial for normal embryonic development (see the Introduction). The half-life of a hybrid mRNA containing FIE3 was previously estimated to be about 50 min (12Riedl A. Jacobs-Lorena M. Mol. Cell. Biol. 1996; 16: 3047-3053Crossref PubMed Scopus (13) Google Scholar). In this study we estimated the half-life of the FIE5-1-containing s-b mRNA and the FIE5-2-containing s-c mRNA to be about 51 and 65 min, respectively. 3J. Ito and M. Jacobs-Lorena, unpublished observations. Thus, each element appears to have similar “strength.” Each element can act independently, and we found no evidence that the destabilizing activity of these elements is additive or synergistic. The estimated half-lives cited above are significantly longer than the 14–6-min half-life reported previously for the endogenous ftzmRNA (8/deleted in proof.Google Scholar). One reason for this difference may be that our measurements started early during embryonic development (from fertilization to 4 h), whereas endogenous ftztranscription occurs between 1.5 and ∼4.5 h. The gradual decrease offtz mRNA half-life from 14 to 6 min between 2.5 and 4 h of development (8/deleted in proof.Google Scholar) suggests that degrading activity increases as embryonic development progresses. Thus, the degrading activity may be low at the very beginning of embryogenesis. Another conceivable reason for the difference in estimated half-lives might be the cytoplasmic localization of the mRNAs (28Mason J.O. Williams G.T. Neuberger M.S. Genes Dev. 1988; 2: 1003-1011Crossref PubMed Scopus (48) Google Scholar, 29Zambetti G. Stein J. Stein G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2683-2687Crossref PubMed Scopus (25) Google Scholar). Endogenousftz mRNA is located in the apical cytoplasm, whereas the distribution of the hybrid mRNAs in the transgenic embryos is unknown (30Fontes A.M. Ito J. Jacobs-Lorena M. Curr. Top. Dev. Biol. 1999; 44: 171-202Crossref PubMed Scopus (14) Google Scholar). Apical localization requires the last 53 nucleotides of the ftz 3′ UTR (31Davis I. Ish-Horowicz D. Cell. 1991; 67: 927-940Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 32Lall S. Francis-Lang H. Flament A. Norvell A. Schüpbach T. Ish-Horowics D. Cell. 1999; 98: 171-180Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), 43 nucleotides of which overlap with FIE3. Deletion of FIE3 (which comprises most of the apical localization sequence) in ftz+aldB-FIE3 did not seem to substantially affect stability when compared with the FIE3-containing ftz+aldB mRNA, suggesting that mRNA stability and localization are independent of each other. The r-abc and s-abc mRNAs both contain the identical 5′-ftz sequence; yet their stability in the early embryo differs dramatically. The main difference between the two mRNAs is the position of the ftz sequences within the mRNA: in the 3′ UTR for r-abc and in the original 5′-position for s-abc. Therefore, the structure and sequence of the RNA elements are not sufficient for destabilizing activity, and position within the mRNA is crucial. Note that when FIE3 was inserted at the same position in the rpA1 3′ UTR as were the FIE5s in r-abc, FIE3 had full destabilizing activity. Thus, FIE3 is active, and FIE5s are inactive when inserted at the identical position of the rpA1mRNA. These results suggest that FIE5s and FIE3 destabilize mRNAs by different mechanisms. This position dependence of the FIE5 elements suggests that translation is required for degradation to occur. Precedents exist for a connection between mRNA stability and translation (30Fontes A.M. Ito J. Jacobs-Lorena M. Curr. Top. Dev. Biol. 1999; 44: 171-202Crossref PubMed Scopus (14) Google Scholar, 33Hennigan A.N. Jacobson A. Mol. Cell. Biol. 1996; 16: 3833-3843Crossref PubMed Google Scholar, 34Oliveira C.C. McCarthy J.E.G. J. Biol. Chem. 1995; 270: 8936-8943Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 35Schiavi S.C. Wellington C.L. Shyu A.-B. Chen C.-Y.A. Greenberg M.E. Belasco J.G. J. Biol. Chem. 1994; 269: 3441-3448Abstract Full Text PDF PubMed Google Scholar, 36Veyrune J.L. Carillo S. Vié A. Blanchard J.M. Oncogene. 1995; 11: 2127-2134PubMed Google Scholar). The suggested dependence of FIE5 activity on mRNA translation is consistent with the results of Edgar et al. (8/deleted in proof.Google Scholar), who reported that general inhibition of embryonic protein synthesis by cycloheximide injection stabilizes the ftzmRNA. However, these results do not rule out the alternative possibility that cycloheximide prevents the synthesis of an unstable protein required for mRNA degradation. The three cis-acting ftz instability elements are likely to act by providing a binding site for a factor or a protein complex that mediates mRNA degradation. A sequence comparison among the three elements and a search for similarity with sequences deposited in data bases did not yield any significant homologies. Binding sites could be recognized as secondary structures rather than primary sequences (37Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar). However, no stable secondary structure that has more than a four-base straight stem or a common secondary structure among the three elements was predicted when a computer program of Zuker (26Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1712) Google Scholar, 38Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288 (presence of 41 additional sgs3-encoded nucleotides at the 5′-end of all): 911-940Crossref PubMed Scopus (3187) Google Scholar) was used to fold these sequences. Moreover, site-directed mutagenesis of certain nucleotides within FIE3 did not alter the destabilizing activity of this element.2 Thus, it is presently unclear how these cis-acting elements are recognized in the embryo. Recently, a protein that binds to the ftz apical localization element was identified (32Lall S. Francis-Lang H. Flament A. Norvell A. Schüpbach T. Ish-Horowics D. Cell. 1999; 98: 171-180Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The identification of proteins that recognize the three instability elements characterized in this and in previous work will bring significant insights to the questions of sequence recognition and mechanism of RNA degradation. We thank Robert Cohen and Kam Cheung (Columbia University) for providing a plasmid containing thesgs3 promoter and the nurse cell-specific enhancer fromhsp26.