A CCHC metal-binding domain in Nanos is essential for translational regulation
1997; Springer Nature; Volume: 16; Issue: 4 Linguagem: Inglês
10.1093/emboj/16.4.834
ISSN1460-2075
AutoresDaniel Curtis, Daniel K. Treiber, Tao Feng, Phillip D. Zamore, James R. Williamson, Ruth Lehmann,
Tópico(s)RNA modifications and cancer
ResumoArticle15 February 1997free access A CCHC metal-binding domain in Nanos is essential for translational regulation Daniel Curtis Daniel Curtis Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Search for more papers by this author Daniel K. Treiber Daniel K. Treiber Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Feng Tao Feng Tao Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Phillip D. Zamore Phillip D. Zamore Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author James R. Williamson James R. Williamson Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Ruth Lehmann Ruth Lehmann Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Search for more papers by this author Daniel Curtis Daniel Curtis Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Search for more papers by this author Daniel K. Treiber Daniel K. Treiber Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Feng Tao Feng Tao Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Phillip D. Zamore Phillip D. Zamore Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author James R. Williamson James R. Williamson Department of Chemistry, Massachusetts Institute of Technology, USA Search for more papers by this author Ruth Lehmann Ruth Lehmann Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA Search for more papers by this author Author Information Daniel Curtis1,3,‡, Daniel K. Treiber2,‡, Feng Tao2,‡, Phillip D. Zamore1,2, James R. Williamson2 and Ruth Lehmann1,4 1Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA, 02142 USA 2Department of Chemistry, Massachusetts Institute of Technology, USA 3Exelixis Pharmaceuticals, Inc., One Kendall Square, Bldg 600, Cambridge, MA, 02139 USA 4Skirball Institute, New York University Medical Center, 540 First Avenue, New York, NY, 10016 USA ‡D.Curtis, D.K.Treiber and F.Tao contributed equally to this work The EMBO Journal (1997)16:834-843https://doi.org/10.1093/emboj/16.4.834 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior–posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex. Introduction In the Drosophila embryo, spatially localized gene expression is critical for the generation of the anterior–posterior body axis. One mechanism essential for achieving localized protein expression is regulated translation of maternal RNA (reviewed in Curtis et al., 1995b). nanos RNA is localized to the posterior pole of the egg during oogenesis, and Nanos protein, translated from this discrete source, diffuses to form a posterior to anterior gradient. Nanos protein then functions to limit the translation of uniformly distributed maternal hunchback RNA in the posterior of the embryo (Tautz and Pfeifle, 1989). hunchback encodes a transcription factor whose graded distribution produces differential expression of target genes along the anterior–posterior axis (Hulskamp et al., 1990). Cis-acting sequences required for translational regulation of hunchback RNA have been mapped to the 3′UTR and are called the Nanos Response Elements (NREs) (Wharton and Struhl, 1991). Mutations in the Nanos protein and mutations in the hunchback NRE both cause ectopic posterior translation of Hunchback protein, producing larvae that completely lack abdominal segments (Tautz and Pfeifle, 1989; Lehmann and Nusslein-Volhard, 1991; Wharton and Struhl, 1991). Nanos-dependent translational repression requires the pumilio gene. In contrast to Nanos, Pumilio protein is present in excess and is distributed throughout the embryo (Barker et al., 1992; Macdonald, 1992). Recently it has been shown that Pumilio protein and a second factor of 55 kDa bind specifically to NRE sequences in embryonic extracts, even in the absence of Nanos protein (Murata and Wharton, 1995). However, the molecular mechanisms that lead to hunchback translational repression, including the role of Nanos, remain unclear. nanos RNAs from several Dipteran species are posteriorly localized in embryos and can substitute functionally for nanos in Drosophila melanogaster (Curtis et al., 1995a). Despite this functional conservation, most of the Nanos protein sequence has diverged. A 72 amino acid C-terminal region, however, is highly conserved among the Nanos proteins and contains an invariant set of eight cysteine and histidine residues. This protein domain is also conserved in a Xenopus gene, Xcat-2, which produces an RNA localized to the vegetal pole of the oocyte (Mosquera et al., 1993). The order of cysteine and histidine residues in the Nanos sequence, along with the established role for many zinc-binding proteins in binding to nucleic acids, suggests the hypothesis that this highly conserved protein domain forms two CCHC zinc-binding sites and that its role in translational regulation includes RNA binding. Since mutants in nanos and in the NREs have the same phenotype, the presence of the CCHC elements suggested the simple model that Nanos is an RNA-binding protein that binds directly to the NREs. To test this possibility, we constructed a series of mutants in the NREs and in Nanos to be used in phenotypic assays in vivo in parallel with RNA binding assays in vitro. Using deletion and point mutants, we show that the C-terminus of Nanos containing the two conserved CCHC motifs is absolutely required for activity, and that both CCHC motifs are essential. The two CCHC motifs are shown to be independent metal-binding elements, each capable of binding Zn(II) or Co(II) ions. Finally, we demonstrate that Nanos binds to RNA with high affinity; however, Nanos alone cannot discriminate between mutant and wild-type NRE RNAs. Results Mutations define the C-terminus as a functional domain of the Nanos protein Previous comparisons between the nanos genes from D.melanogaster and three other Dipteran species revealed high conservation in a 72 amino acid C-terminal domain, but little conservation elsewhere among these ∼400 amino acid proteins (Curtis et al., 1995a). The heterologous Nanos proteins were functional in D.melanogaster, suggesting that much of the Nanos protein activity resides in the C-terminal domain. To test this hypothesis we deleted large portions of the Nanos protein sequence and assayed the mutants for function in an RNA injection rescue assay. In this assay, injection of in vitro-transcribed wild-type nanos RNA into eggs laid by nanos mutant mothers leads to complete rescue of abdominal segmentation, and rescued larvae can hatch and develop into fertile adults (Wang and Lehmann, 1991; Curtis et al., 1995a). Deletion of amino acids 50–218 (Δ50–218) had a strong effect on Nanos function, Δ218–287 had no detectable effect, and Δ288–314 had a moderate effect (Figure 1). Limited amino acid sequence similarity between the Dipteran Nanos proteins is found in the 50–218 and 288–314 regions, while no similarity is found in the 218–287 region (Figure 1). The D.melanogaster N-terminal 1–50 amino acid region is not present in other Nanos proteins, which can substitute for D.melanogaster Nanos, suggesting that this region is not critical for function. Together, these observations show that no single region outside the C-terminal 87 amino acid region is absolutely required for Nanos protein function. However, when RNAs encoding the C-terminal 87, 99 or 114 amino acids of Nanos were injected (Δ2–314, Δ2–301, Δ2–287), no rescuing activity was detected. Nanos protein was synthesized from these deletion RNAs, as detected by in vitro translation of injection RNAs and by α-Nanos antibody staining of injected embryos (Figure 1). Thus, neither the C-terminal domain alone, nor the C-terminal domain together with the adjacent conserved asparagine- and lysine-rich region (amino acids 288–314), are sufficient for Nanos activity in vivo. Structures required for Nanos function therefore reside in the 50–218 and 288–314 regions of the Nanos protein, and removal of both regions, as in Δ2–314, eliminates detectable Nanos activity. Figure 1.(A) Conserved regions of Nanos, based on interspecific comparisons, are indicated by open boxes (Curtis et al., 1995a). The protein sequence is represented to scale. Only the central 11-residue region (open box) and the C-terminal CCHC domain are conserved in primary sequence. S/T, a region rich in serine and threonine residues. N/K, a region rich in asparagine and lysine residues. (B) Deletions drawn to scale, with relative degree of rescue in the RNA injection assay indicated at right. (C) Sequence of the C-terminal 99 amino acids of Nanos, with positions of the nanos point mutations and conserved His and Cys residues (black dots) indicated. nosRD changes nt 1943 from G to A, changing a TGC cysteine codon to a TAC tyrosine codon. nosL7 deletes nt 2009–2029, inclusive, eliminating seven codons (ITMEDAI). nosRW changes nt 2020 from G to A, altering a GAT aspartic acid codon to an AAT asparagine codon. (D) RNA injection rescue results. 'Overall rescue' is the percentage of larvae with any rescue, i.e. 1–8 segments, out of all developing larvae. 'Strong rescue' is the percentage of larvae with 5–8 segments. n = number of larvae scored (results from separate trials were consistent and were pooled); # trials = number of independent RNA preparations tested. Nos protein staining: 4(+) indicate staining intensity roughly equivalent to that observed at the posterior pole of wild-type uninjected embryos. Staining of the injected embryos was centered around the site of injection, with diffuse staining throughout the egg. 1(+) indicates that staining was weak and most easily detected in occasional pole cells that stained positively. Such staining was never observed in uninjected embryos. Download figure Download PowerPoint To further identify regions of the Nanos protein critical for its function, we analyzed four mutant alleles of nanos (nos). One allele, nosRC, disrupts the splice donor site of the first nanos intron [nucleotide 734 G to A in the sequence of Wang and Lehmann (1991)]. This allele fails to produce nanos RNA or protein, and is therefore a null mutation (Wang et al., 1994). The other three alleles, nosL7, nosRW and nosRD all affect sequences in the C-terminal region of the Nanos protein (Figure 1). nosL7, which behaves as a strong allele for abdominal segmentation but retains oogenesis function, is an in-frame deletion of seven amino acids (I376–I382) just outside of the second CCHC domain, while nosRW, a weak allele that produces embryos with variable numbers of abdominal segments, is an aspartic acid to asparagine change affecting one of the residues deleted in nosL7 (D380N). nosRD, like nosRC, is a strong allele, eliminating both the abdominal segmentation and oogenesis functions of nanos (Wang et al., 1994). The sequence CX2C is found in many zinc-binding proteins, and is present in both of the conserved Nanos CCHC motifs. nosRD changes the first cysteine residue in the second CX2C motif to tyrosine (C354Y). We changed the first cysteine in motif 1, C319, to tyrosine by in vitro mutagenesis, a change analogous to the nosRD C354Y mutation in motif 2. When tested by RNA injection assay, in vitro-transcribed C319Y RNA had no Nanos rescuing activity (Figure 1). Thus, both CX2C motifs are essential for Nanos protein function. The Nanos C-terminus contains two consecutive CCHC metal-binding sites The results described above, together with the resemblance of Nanos to known zinc finger proteins, suggest the presence of two zinc-binding sites in Nanos. To assess the metal-binding properties of Nanos, wild-type and mutant C-terminal 99 amino acid Nanos polypeptides (amino acids 302–401; Nos99) were purified from Escherichia coli overexpression strains and assayed for Co(II) and Zn(II) binding. Co(II) can structurally replace Zn(II) in many metalloproteins, but, unlike Zn(II), Co(II)-bound forms of proteins have pronounced absorbance peaks in the UV and visible regions of the spectrum that facilitate quantitative analysis. In addition, the visible absorbance spectrum of Co(II) is sensitive to the ligand environment and thus helps to establish the identity of coordinating side chains (Shi et al., 1993). Addition of Co(II) to the Nanos apoprotein (Nos99, Figure 1) caused dramatic UV and visible absorbance changes with peaks at 313, 640 and 713 nm (Figure 2A), establishing that Nos99 contains a metal-binding domain. Furthermore, these absorbance peaks are characteristic of CCHC coordination (Green and Berg, 1989; Krizek et al., 1991; Shi et al., 1993). The simplest interpretation of this result is that Nos99 contains two consecutive CCHC metal-binding units. More complicated model structures however, including the interleaved ligand arrangements identified in the RING finger (Barlow et al., 1994) and protein kinase C (Hommel et al., 1994), remained possible. To distinguish between the models, a smaller polypeptide containing only the C-terminal 54 amino acids of Nanos (amino acids 347–401, Nos54) was prepared. Nos54, which contains only the second CCHC motif, also binds Co(II), and the visible spectrum qualitatively resembles that of the Nos99 complex (Figure 2). Nos54 thus forms an independent, stable CCHC metal-binding domain, strongly supporting the consecutive CCHC model. Figure 2.Absorbance spectra of Co(II)-complexed Nos polypeptides. (A) UV/visible spectrum of Nos99 complexed with Co(II). (B) Visible spectra of Nos54 and Nos99 mutants. The Nos99 wild-type spectrum is included for reference. Protein concentrations were 10–80 μM and Co(II) was present in saturating concentrations. The free protein spectra have been subtracted, and extinction coefficients were calculated by dividing the corrected absorbance values by the protein concentration. Download figure Download PowerPoint The consecutive CCHC model was tested further by examining the Co(II) spectra of mutant Nos99 polypeptides. The model predicts that Nos99C319Y and Nos99C354Y should lack only the first or second metal coordination site, respectively. Metal binding to the double mutant (Nos99C319,354Y) should be abolished. As shown in Figure 2, both single mutants retain Co(II) binding activity, whereas the double mutant does not. The Co(II) complex spectra of the single Cys mutants are consistent with CCHC coordination. Like Nos54, the mutants exhibit reduced extinction coefficients relative to Nos99, suggesting reduced Co(II):protein ratios relative to wild-type. These results strongly support a consecutive arrangement of independent CCHC metal-binding sites. Since the double mutant is completely inactive in Co(II) binding, non-native metal coordination is not observed. To determine the stoichiometry of metal binding, apo-Nos polypeptides were titrated quantitatively with Co(II). The endpoints of stoichiometric titrations establish that Nos99 binds two molar equivalents of Co(II), while Nos54, Nos99C319Y and Nos99C354Y each bind one equivalent (Figure 3). We conclude that the C-terminal domain of Nanos contains two independent metal-binding sites, both of which are required for Nanos activity in vivo. Figure 3.Co(II) binding stoichiometry of the Nanos polypeptides: (A) Nos99; (B) Nos99C319Y; (C) Nos99C354Y and (D) Nos54. Co(II) was titrated into Nos polypeptide samples and the absorbance increase at 313 nm (A and D) or 640 nm (B and C) was recorded. The fraction of sites bound was calculated by normalizing the absorbance changes to the maximum value. The x axes represent the ratio of Co(II) concentration to protein concentration. Download figure Download PowerPoint Although we have not determined the metal coordinated by Nanos protein in vivo, Zn(II) is a likely candidate. Zn(II) binding by Nanos protein in vitro was measured indirectly by competition analysis (Figure 4). Nos polypeptides were reconstituted with excess Co(II), and the decrease in absorbance at 313 nm or 640 nm was recorded as a function of Zn(II) concentration. In all cases, competition with Zn(II) was stoichiometric, and the metal:protein ratios determined by Co(II) titration (Figure 3) were recapitulated. Since Zn(II) binding was stoichiometric even in the presence of a 10-fold excess of Co(II), we estimate that Zn(II) binding is preferred over Co(II) by at least three orders of magnitude. Co(II) binding is stoichiometric at ∼10 μM protein, the lowest concentration tested, indicating that the upper limit for the Kd of Co(II) is 1 μM and that Zn(II) binds with sub-nanomolar affinity. Specificity for Zn(II) over Co(II) is common and may result from losses in ligand field stabilization energy that occur when Co(II) changes from an octahedral aqueous species to a tetrahedral species in proteins (Lippard and Berg, 1994). Figure 4.Zn(II) binding stoichiometry of Nos polypeptides. (A) Nos99; (B) Nos99C319Y; (C) Nos99C354Y; (D) Nos54. Zn(II) was titrated into Nos polypeptide samples that were equilibrated with 11 molar equivalents of Co(II). Zn(II) binding was detected by a decrease in Co(II)-dependent absorption at 313 nm (A and D), or 640 nm (B and C). Data calculations were performed as for Figure 3. Download figure Download PowerPoint Characterization of Zn(II) binding to the Nanos CCHC domain by NMR spectroscopy NMR studies have shown that zinc promotes folding of classical and CCHC-type zinc fingers (Parraga et al., 1988; Lee et al., 1989; South et al., 1989), and one-dimensional 1H-NMR spectroscopy of the C-terminal Nos99 polypeptide provides further evidence for Zn(II) coordination. Addition of Zn(II) to the Nos99 apoprotein results in increased resonance dispersions indicative of structure formation. The chemical shift changes are most distinctive for some aliphatic protons (data not shown) and for His-H2 and -H4 protons (Figure 5A). The His-H2 and -H4 protons are useful indicators of Zn(II) coordination. In the apoprotein, the three His-H2 proton resonances are present as two overlapped peaks at 8.3 p.p.m. and a single peak at 8.2 p.p.m. Two of these peaks shift to 7.7 and 7.8 p.p.m. upon addition of Zn(II) (Figure 5A, arrows) and attain maximum intensity at a Zn(II):protein ratio of 2:1, as expected for the binding of two molar equivalents of zinc. The appearance of these two peaks is concurrent, suggesting that the CCHC sites have comparable Zn(II) affinities or, alternatively, that binding is highly cooperative. The third His-H2 proton only forms detectable metal complexes when excess Zn(II) is added (Figure 5A, asterisk), suggestive of non-specific binding. Zn(II) titrations were also performed on the Nos54 and Nos99C354Y apoproteins. The spectra of the apoproteins and the titration end-points are shown in Figure 5B. For Nos54, the His365 proton peak at 7.85 p.p.m. reaches maximum intensity at a Zn(II):protein ratio of 1:1. Similarly, for Nos99C354Y a His proton peak at 7.8 p.p.m. reached a maximum at a Zn(II):protein ratio of 1:1 (Figure 5B, arrow). The two additional His peaks, most likely representing His318 and His365, do not shift to the 7.8 p.p.m. region. The His protons shifted to ∼7.8 p.p.m. in Nos54 and Nos99C354Y likely correspond to the 7.7 and 7.8 p.p.m. His protons in the Nos99 spectrum. Figure 5.Aromatic proton region from 1H-NMR spectra of Nos polypeptides. (A) Zn(II) titration of Nos99 at pH 6.4. Molar ratios of Zn(II) to protein are shown at left. Arrows indicate the two H2 protons of the His residues coordinated to Zn(II); these increase in intensity as Zn(II) is titrated. Filled squares and asterisks indicate the H2 and H4 protons from a third His, most likely His318 (see text). This His only binds Zn(II) after binding to the other two His residues is saturated at Zn(II):polypeptide ratios of >2. (B) Spectra of the apo-Nos polypeptides compared with spectra after addition of 2 (Nos99) or 1 (Nos54 and Nos99C354Y) molar equivalents of Zn(II). Arrows indicate positions of protons from Zn(II)-liganded His residues. (C) pH titration of apo-Nos99. The chemical shifts of His-H2 and -H4 protons are pH sensitive in the absence of Zn(II). (D) pH titration of Nos99 reconstituted with 2.4 molar equivalents of Zn(II). The chemical shifts of H2 and H4 protons from two His residues are pH insensitive (arrows), while those from the third His are highly sensitive (filled squares). Asterisks indicate H2 and H4 protons from the third His that are pH-insensitive due to non-specific Zn(II) coordination. Download figure Download PowerPoint To confirm the assignment of the 7.7 and 7.8 p.p.m. proton shifts to His–Zn(II) complexes, pH titration experiments were performed (Figure 5C and D His is complexed to metal, the chemical shifts of its H2 and H4 protons are insensitive to moderate changes in pH, while an uncomplexed His will demonstrate pH-dependent chemical shifts with the transition midpoint near the pKa of free His (∼6.0) (Parraga et al., 1990). A pH titration of apoNos99 shows that the His-H2 proton (peaks near 8.5 p.p.m.) move upfield with increasing pH (Figure 5C). A similar pH titration for Zn(II)-reconstituted Nos99 is shown in Figure 5D. In this sample the His peaks at 7.7 and 7.8 p.p.m. are pH-insensitive, indicating that a bound metal is blocking protonation. In contrast, the third His proton chemical shift (Figure 5D, filled squares) shows a strong pH-dependence similar to that observed for the His protons in the apo-Nos99 sample (Figure 5C). In addition, the peak resulting from the third His–Zn(II) complex (asterisks in Figure 5D) is relatively pH sensitive, providing further evidence against a role for the third His (likely His318) in zinc coordination. We conclude from these studies that Nos99 contains two histidine residues complexed to Zn(II), and that these contribute to two consecutive, independent CCHC zinc-binding domains. NRE mutations disrupt translational repression Since many zinc-binding proteins function by binding to nucleic acids, and because of the known role of the NRE sequence in Nanos-dependent regulation of hunchback translation, we tested for the ability of Nanos proteins to bind to NRE-containing RNAs. In preliminary experiments we found that C-terminal Nanos polypeptides bind to RNA in vitro in UV crosslinking assays (data not shown). The NRE target sequence for Nanos-dependent translational repression was previously defined as a 32 nucleotide repeat present in the hunchback and bicoid RNAs, in which only 11 out of the 32 nucleotide positions are conserved (Wharton and Struhl, 1991). In order to determine the importance of the conserved nucleotide sequences, and to create sequences suitable for testing the specificity of Nanos RNA binding in vitro, we mutated the NREs and tested their ability to repress translation of hunchback mRNA in transgenic animals. When all six conserved guanosines in the NRE core sequences are changed to uracil (G1–6U, Figure 6), the transgene produces a dominant female sterile phenotype in which all abdominal segmentation is repressed, a phenotype indistinguishable from the nanos mutant phenotype. To test for residual NRE function we raised the effective Nanos protein concentration in G1–6U transgenic embryos in two ways, by injection of in vitro-transcribed nanos RNA, and by the introduction of the torso or torsolike mutations (see Materials and methods). In neither experiment did we observe any rescue of the G1–6U dominant phenotype. G1–6U thus behaves as a complete loss of function NRE mutation. Figure 6.Mutations in the hunchback NRE sequences and their effects. A wild-type hunchback reporter transgene is fully regulated, resulting in larvae with the wild-type cuticle pattern drawn at right (anterior to left) (Wharton and Struhl, 1991). Mutation of all six conserved NRE guanosine residues to uracil (G1–6U) results in no translational repression, and a mutant cuticle pattern identical to that seen in a complete deletion of the NREs (Wharton and Struhl, 1991). The G1–3U mutation targeting the first NRE results in a much stronger mutant phenotype than G4–6U, which affects the second NRE. 'Phenotype' illustrates average number of abdominal segments formed by embryos from several independent transgenic lines. Download figure Download PowerPoint Mutations in the first (G1–3U) and second (G4–6U) NRE sequences were also tested independently in the same transgenic assay (Figure 6). G1–3U mutant transgenes produced a dominant female sterile phenotype, with all embryos developing into larvae with two to five segments. G4–6U transgenes did not result in female sterility, although examination of the larvae from transgenic females showed that the majority had mild defects in one or two abdominal segments (Figure 6). Thus, both NREs contribute to translational repression in vivo, but the first NRE contributes much more activity than the second. While no sequence similarity between NREs is found outside the conserved 11 nucleotide motifs, these results, in agreement with those of Murata and Wharton (1995) suggest that other non-conserved sequences within the 32 nucleotide NRE influence Nanos-dependent regulation. Nanos binds RNA non-specifically To test for RNA binding specificity in vitro, Nanos proteins were prepared and assayed in filter binding assays. Full-length Nanos was expressed as a maltose-binding protein fusion (MBPNos) and purified under native conditions by amylose and heparin affinity chromatography. Nos99 was prepared under denaturing conditions as described above, and reconstituted with Zn(II). RNA substrates tested include the wild-type NRE fragment (DX), and three RNAs with no NRE activity in vivo: DX[G1–6U], an adjacent fragment of the hunchback 3′UTR (XA), and a fragment of the α-tubulin 3′UTR (TUB) (this work, and Wharton and Struhl, 1991). In filter binding assays we observed that MBPNos and Nos99 bind tightly to the DX RNA (Kd ∼50 nM, data not shown). The Nanos RNA binding activity therefore resides in the C-terminal Zn binding domain. To address binding specificity, a filter retention assay was used in which the binding reaction contains two RNAs, both in excess concentration over Nanos protein (Bartel et al., 1991). The ratio of the RNAs retained on the filter reflects the relative affinity (i.e. ratio of the Kd values) of the protein for the two RNAs. The data presented in Table I illustrate that MBPNos and Nos99 behave indistinguishably in the binding assay. MBPNos and Nos99 proteins show modest binding preferences for DX over TUB or XA RNAs (9- and ∼3-fold, respectively). However, we find that neither protein discriminates between the wild-type DX and DX[G1–6U] RNAs. Since the DX[G1–6U] mutant is completely inactive in vivo, it should affect the binding of proteins that interact specifically with the core NRE sequences. These results suggest that Nanos RNA binding is only modestly sequence specific and does not involve direct recognition of the conserved G residues in the NRE sequences. Table 1. Competition RNA binding assays Protein RNAs Ratio n MBPNos DX/TUB 9.1 ± 4.4 8 MBPNos DX/XA 4.5 ± 1.2 7 MBPNos DX/DX[G1–6U] 1.2 ± 0.3 3 Nos99 DX/TUB 9.1 ± 2.5 3 Nos99 DX/XA 2.5 ± 0 2 Nos99 DX/DX[G1–6U] 0.7 ± 0.1 3 apo-Nos99 DX/TUB 1.6 ± 0.1 2 apo-Nos99 DX/XA 0.8 ± 0.1 2 apo-Nos99 DX/DX[G1–6U] 0.7 ± 0 2
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