FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2033200059> ?p ?o ?g. }

• W2033200059 endingPage "8894" @default.
• W2033200059 startingPage "8883" @default.
• W2033200059 abstract "Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9–13-nucleotide window. This sequence is remarkably abundant in the 3′-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation. Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9–13-nucleotide window. This sequence is remarkably abundant in the 3′-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation. Embryogenesis is the process by which a fertilized oocyte transforms into a multicellular organism. Although the zygote contains all of the information required for development, zygotic DNA alone is not sufficient to drive patterning. Somatic cell nuclear transfer experiments, like those used to clone Dolly the sheep, demonstrate that maternal factors present in the oocyte cytoplasm are needed for the initiation of development (1Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Nature. 1997; 385: 810-813Crossref PubMed Scopus (4038) Google Scholar). These maternal factors are proteins and quiescent mRNAs (2Seydoux G. Braun R.E. Cell. 2006; 127: 891-904Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar); they coordinate early development prior to the onset of zygotic transcription. In the nematode worm Caenorhabiditis elegans, polarization of the body axes occurs after fertilization and requires several highly conserved maternal factors termed PAR proteins (3Etemad-Moghadam B. Guo S. Kemphues K.J. Cell. 1995; 83: 743-752Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 4Goldstein B. Hird S.N. Development. 1996; 122: 1467-1474Crossref PubMed Google Scholar, 5Guo S. Kemphues K.J. Cell. 1995; 81: 611-620Abstract Full Text PDF PubMed Scopus (884) Google Scholar, 6Kemphues K.J. Priess J.R. Morton D.G. Cheng N.S. Cell. 1988; 52: 311-320Abstract Full Text PDF PubMed Scopus (684) Google Scholar, 7Kirby C. Kusch M. Kemphues K. Dev. Biol. 1990; 142: 203-215Crossref PubMed Scopus (119) Google Scholar, 8Levitan D.J. Boyd L. Mello C.C. Kemphues K.J. Stinchcomb D.T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6108-6112Crossref PubMed Scopus (75) Google Scholar, 9Tabuse Y. Izumi Y. Piano F. Kemphues K.J. Miwa J. Ohno S. Development. 1998; 125: 3607-3614Crossref PubMed Google Scholar, 10Wallenfang M.R. Seydoux G. Nature. 2000; 408: 89-92Crossref PubMed Scopus (149) Google Scholar). Prior to fertilization, these proteins are uniformly distributed in the cytoplasm. Once the sperm penetrates the oocyte, they localize to opposing cortical domains in a process that requires microtubules derived from the asters of the sperm pronucleus. The PAR network coordinates asymmetric translation of several cell signaling proteins (11Mello C.C. Draper B.W. Priess J.R. Cell. 1994; 77: 95-106Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 12Thorpe C.J. Schlesinger A. Carter J.C. Bowerman B. Cell. 1997; 90: 695-705Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar) (glp-1, apx-1, mom-2, and mom-5) and transcription factors (13Maduro M.F. Kasmir J.J. Zhu J. Rothman J.H. Dev. Biol. 2005; 285: 510-523Crossref PubMed Scopus (73) Google Scholar) (skn-1, pal-1, and pop-1) encoded by maternal mRNAs present throughout the 1-cell embryo. The PAR proteins are thought to locally deactivate maternal RNA-binding proteins thereby modulating the stability or translation efficiency of maternal mRNAs. Consistent with this hypothesis, posterior localization of PAR-1 promotes anterior localization of two putative RNA-binding proteins, MEX-5 and MEX-6 (14Schubert C.M. Lin R. de Vries C.J. Plasterk R.H. Priess J.R. Mol. Cell. 2000; 5: 671-682Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 15Cuenca A.A. Schetter A. Aceto D. Kemphues K. Seydoux G. Development. 2003; 130: 1255-1265Crossref PubMed Scopus (219) Google Scholar). Although these proteins are 70% identical, their contributions to development only partially overlap. Disruption of mex-5 causes embryonic death with a terminal phenotype that includes proliferation of muscle (MEX = muscle excess). In contrast, deletion of mex-6 does not affect viability. Two major roles in development have been attributed to MEX-5. First, it controls segregation of the germline from the soma by activating zif-1, which promotes anterior turnover of three germline-specific maternal proteins (POS-1, PIE-1, and MEX-1) (16DeRenzo C. Reese K.J. Seydoux G. Nature. 2003; 424: 685-689Crossref PubMed Scopus (136) Google Scholar). The overall result of this pathway is a gradient of MEX-5/6 from anterior to posterior and an opposing gradient of POS-1, PIE-1, and MEX-1 (Fig. 1A). Second, MEX-5 plays a relatively uncharacterized role in maintaining PAR polarity via a feedback loop with PAR-1 (15Cuenca A.A. Schetter A. Aceto D. Kemphues K. Seydoux G. Development. 2003; 130: 1255-1265Crossref PubMed Scopus (219) Google Scholar). It is not yet clear if the two roles are linked at the molecular level. Additionally, there may be other roles for MEX-5 that have not yet been described. For example, residual posterior MEX-5 accumulates on the posterior centrosome and in P-granules, RNA-rich bodies that segregate with and determine the germline lineage (14Schubert C.M. Lin R. de Vries C.J. Plasterk R.H. Priess J.R. Mol. Cell. 2000; 5: 671-682Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 15Cuenca A.A. Schetter A. Aceto D. Kemphues K. Seydoux G. Development. 2003; 130: 1255-1265Crossref PubMed Scopus (219) Google Scholar). The functional ramifications of this localization are not known. MEX-5, MEX-6, POS-1, PIE-1, and MEX-1 are all CCCH-type tandem zinc finger proteins (Fig. 1B, hereafter TZF). 2The abbreviations used are: TZF, CCCH-type tandem zinc finger; TTP, tristetraprolin; UTR, untranslated region; ARE, AU-rich element; TNF, tumor necrosis factor; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; FP, fluorescence polarization; TCR, temporal control region; RNAi, RNA interference; GFP, green fluorescent protein; DIC, differential interference contrast.2The abbreviations used are: TZF, CCCH-type tandem zinc finger; TTP, tristetraprolin; UTR, untranslated region; ARE, AU-rich element; TNF, tumor necrosis factor; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; FP, fluorescence polarization; TCR, temporal control region; RNAi, RNA interference; GFP, green fluorescent protein; DIC, differential interference contrast. This class is typified by tristetraprolin (TTP), a mammalian protein involved in regulating inflammation response by destabilizing TNF-α transcripts (17Varnum B.C. Ma Q.F. Chi T.H. Fletcher B. Herschman H.R. Mol. Cell. Biol. 1991; 11: 1754-1758Crossref PubMed Scopus (149) Google Scholar, 18Taylor G.A. Carballo E. Lee D.M. Lai W.S. Thompson M.J. Patel D.D. Schenkman D.I. Gilkeson G.S. Broxmeyer H.E. Haynes B.F. Blackshear P.J. Immunity. 1996; 4: 445-454Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 19Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 20Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (630) Google Scholar). The expression pattern of several key maternal transcripts is perturbed in TZF mutants leading to the hypothesis that they directly regulate maternal mRNA stability or translation efficiency (14Schubert C.M. Lin R. de Vries C.J. Plasterk R.H. Priess J.R. Mol. Cell. 2000; 5: 671-682Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 21D'Agostino I. Merritt C. Chen P.L. Seydoux G. Subramaniam K. Dev. Biol. 2006; 292: 244-252Crossref PubMed Scopus (60) Google Scholar, 22Ogura K.I. Kishimoto N. Mitani S. Gengyo-Ando K. Kohara Y. Development. 2003; 130: 2495-2503Crossref PubMed Scopus (74) Google Scholar, 23Reese K.J. Dunn M.A. Waddle J.A. Seydoux G. Mol. Cell. 2000; 6: 445-455Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 24Tabara H. Hill R.J. Mello C.C. Priess J.R. Kohara Y. Development. 1999; 126: 1-11Crossref PubMed Google Scholar). If so, then the network of maternal RNA regulation in the embryo may be governed by differences in the RNA-binding specificity of each protein. Consistent with this model, TTP is an exquisitely specific RNA-binding protein; it recognizes nonameric UUAUUUAUU sequences present in the 3′-untranslated region (UTR) of its targets (19Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar, 20Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (630) Google Scholar, 25Lai W.S. Parker J.S. Grissom S.F. Stumpo D.J. Blackshear P.J. Mol. Cell. Biol. 2006; 26: 9196-9208Crossref PubMed Scopus (182) Google Scholar, 26Brewer B.Y. Malicka J. Blackshear P.J. Wilson G.M. J. Biol. Chem. 2004; 279: 27870-27877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). An NMR structure of the mammalian TTP homolog ERF-2 (also known as Tis11D) demonstrates that each finger individually recognizes a UAUU repeat (27Hudson B.P. Martinez-Yamout M.A. Dyson H.J. Wright P.E. Nat. Struct. Mol. Biol. 2004; 11: 257-264Crossref PubMed Scopus (282) Google Scholar). In contrast, an in vitro interaction between any of the TZF proteins from C. elegans and RNA has not been demonstrated, and as such their mRNA target specificity has not been explored. MEX-5 and MEX-6 diverge from TTP in a few notable ways (Fig. 1B): (i) nine amino acids rather than eight separate the first two cysteines in each zinc finger, (ii) the spacing between fingers is lengthened, and (iii) several highly conserved amino acids that contribute to RNA binding in mammalian TZF proteins are not conserved in MEX-5 and MEX-6. These differences could impact the ability of MEX-5 and MEX-6 to bind to RNA. Moreover, MEX-5 has been shown to interact with ZIF-1 protein in a yeast two-hybrid assay (16DeRenzo C. Reese K.J. Seydoux G. Nature. 2003; 424: 685-689Crossref PubMed Scopus (136) Google Scholar), suggesting that it may not regulate this factor at the RNA level. We set out to describe the RNA-binding properties of MEX-5 to probe its role in patterning the anterior-posterior axis. Protein Expression Constructs—Fragments of mex-5 and mex-6 containing the TZF domain (amino acids 236–350 and 250–400, respectively) were amplified from ORFeome clones (Open Biosystems) and subcloned into the vector pMal-c (New England Biolabs). Mutations of pMal-MEX-5-(236–350) were prepared by site-directed mutagenesis using QuikChange (Stratagene). Purification of Recombinant Proteins—TZF domains from MEX-5, MEX-6, and mutants thereof were expressed and purified from Escherichia coli JM109 cells as C-terminal fusions to maltose-binding protein. Liquid cultures grown at 37 °C were induced in mid-log phase with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. Zinc acetate was added to a final concentration of 100 μm at the time of induction. Harvested cells were resuspended in lysis buffer (50 mm Tris, pH 8.8, 200 mm NaCl, 2 mm dithiothreitol, EDTA-free protease inhibitor tablet (Roche Applied Science), 100 μm Zn(OAc)2) and lysed by sonication. Soluble protein was purified over an amylose column (New England Biolabs). Fractions containing the fusion protein were pooled and dialyzed into Q buffer (50 mm Tris, pH 8.8, 20 mm NaCl, 2 mm dithiothreitol, and 100 μm Zn(OAc)2) and then further purified over a Hi-trap Q HP column (GE Healthcare). Final purification was achieved by combining fractions containing the protein and dialyzing them into S buffer (50 mm MOPS, pH 6.0, 20 mm NaCl, 2 mm dithiothreitol, 100 μm Zn(OAc)2) before running them over a Hi-trap S HP column (GE Healthcare). Care was taken to minimize the amount of time the protein was exposed to pH 6.0 buffer. Pure fractions as determined by Coomassie-stained SDS-PAGE were combined and dialyzed into storage buffer (20 mm Tris, pH 8.0, 20 mm NaCl, 100 μm Zn(OAc)2, 2 mm dithiothreitol). After dialysis, the protein concentration was determined using Beers law by measuring absorbance at 280 nm and a calculated extinction coefficient determined using the ProtParam server (28Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3429) Google Scholar). The protein was concentrated to ∼50 μm before storage at 4 °C. Analytical Gel Filtration Chromatography—A Superdex 200 10/300 GL column (10 × 300 mm, GE Healthcare) was used to determine the apparent molecular weight of MBP-MEX-5-(236–350) and its mutants. The column was equilibrated on an AKTA FPLC with 2 column volumes of filtration buffer (50 mm Tris, pH 8.0, 300 mm NaCl) at a flow rate of 0.5 ml min-1 prior to loading the protein sample. Approximately 50 μl of sample (17 μm) was loaded on to the column and eluted with 1.5 column volumes of filtration buffer. Retention time was determined in relation to standards (Bio-Rad). RNA Labeling Protocol—All of the RNA sequences used in this work were prepared by chemical synthesis and deprotected/lyophylized as the manufacturer directed (Dharmacon or Integrated DNA technologies). Lyophilized samples were resuspended in 300 μl of TE buffer, pH 8.0, and the concentration was measured by determining the absorbance at 260 nm using a calculated extinction coefficient based on the nucleotide content. Fluorescein 5-thiosemicarbazide (Invitrogen) was used to 3′-end label each RNA via the method of Reines and Cantor (29Reines S.A. Cantor C.R. Nucleic Acids Res. 1974; 1: 767-786Crossref PubMed Scopus (38) Google Scholar). A typical 50-μl reaction consisted of 0.5 nmol of RNA, 100 mm NaOAc, pH 5.1, and 5 nmol of NaIO4. After a 90-min incubation at room temperature, the sample was ethanol precipitated with 1 μl of RNase-free glycogen (Invitrogen 20 μg/μl), 5 m NaCl (1/20 the volume), and 2 volumes of 100% ice-cold ethanol. The resulting pellet was resuspended in 50 μl of 100 mm NaOAc, pH 5.1, containing 1 mm fluorescein-5-thiosemicarbazide. This reaction was incubated overnight at 4 °C and unreacted label was removed using a Roche G-25 spin column. The labeling efficiency was determined by calculating the ratio of fluorescein absorbance at 490 nm to RNA-fluorescein absorbance at 260 nm. Typical efficiencies were 60–80%. Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays were used to measure the binding activity of recombinant MEX-5 and MEX-6 to fluorescein-labeled RNA oligonucleotides. Typical reactions consisted of 2–4 nm labeled RNA equilibrated with varying concentrations of protein in equilibration buffer for 3 h. Equilibration buffer is 0.01% IGEPAL CA630 (a mild detergent used to prevent adhesion of protein and RNA to tubes, microplates, and gel wells), 0.01 mg/ml tRNA (a polyanionic nonspecific binding inhibitor), 10 mm Tris, pH 8.0, 100 μm Zn(OAc)2, and 100 mm NaCl. The RNA was heated to 60 °C and allowed to cool to room temperature before use. Immediately prior to loading, one-fifth volume of 30% (v/v) glycerol, 0.01% (w/v) bromcresol green was added to each reaction as a dye marker. A 40-μl sample of each reaction (100 μl total) was loaded onto a 1% agarose gel (EMD Biosciences, some lot to lot variability was observed) in 1× TB buffer. The gels were run for 40 min at 120 volts then immediately scanned using a fluor-imager (Fujifilm FLA-5000) with a blue laser at 473 nm. The fluorescence intensity of unbound RNA was determined as a function of protein concentration using ImageGauge software. The data were fit to a sigmoidal dose-response function (Equation 1) to determine the half-maximal saturation point (Kd,app),ϕ=b+(m-b)11+(Kd,appP)n(Eq.1) where ϕ is the fluorescence intensity, m is the maximal signal, b is the minimal signal, P is the protein concentration, and n is the apparent Hill coefficient. It is important to note that Kd,app is not equivalent to the thermodynamic equilibrium dissociation constant for RNA sequences that contain multiple overlapping binding sites. In all cases, the reported value is the average of at least three experiments and the reported error is the standard deviation. Competition assays were performed as above except a constant concentration of subsaturating recombinant MEX-5 was used in the equilibration, whereas varying concentrations of unlabeled competitor RNA were added to the reactions. The apparent dissociation constant of the competitor RNA was determined by a fit of the data to a quadratic solution of the Lin and Riggs equation (30Lin S.Y. Riggs A.D. J. Mol. Biol. 1972; 72: 671-690Crossref PubMed Scopus (224) Google Scholar, 31Weeks K.M. Crothers D.M. Biochemistry. 1992; 31: 10281-10287Crossref PubMed Scopus (122) Google Scholar) as described (32Ryder S.P. Williamson J.R. RNA (N.Y.). 2004; 10: 1449-1458Crossref PubMed Scopus (74) Google Scholar). Fluorescence Polarization Assays—Equilibration reactions (100 μl volume) were set up using the same conditions as the electrophoretic mobility shift experiments above in 96-well black plates (Greiner). The apparent fluorescence polarization was determined using a Victor 3 plate reader (PerkinElmer Life Sciences) equipped with fluorescein-sensitive filters and polarizers. A total of five reads were measured for each experiment and the average and standard deviation of the millipolarization value (mP) were calculated for each protein concentration. The data were fit to Equation 1 (where ϕ represents polarization rather than intensity) to extract the apparent dissociation constant. The reported value is the average of at least three experiments and the error is the S.D. Stoichiometric binding experiments were performed as above except the reactions were supplemented with unlabeled RNA to a final concentration of 1.5 μm. The elevated concentration of RNA enables determination of the apparent stoichiometry by measuring the equivalent saturation point. This value was estimated by plotting polarization as a function of molar equivalents of protein to RNA and performing linear fits to pre- and post-saturation data. The equivalence point was determined by the intersection point of the two lines, and separately by a fit of the data to the quadratic equation as described (33Rambo R.P. Doudna J.A. Biochemistry. 2004; 43: 6486-6497Crossref PubMed Scopus (57) Google Scholar). UTR Sequence Analysis—C. elegans 3′-UTRs were retrieved from Wormbase release WS165. To determine the frequency of each possible octamer sequence in 3′-UTR-space, a Ruby script was written to enumerate each possible octamer and send it to the pattern searching tool PATSCAN (34Dsouza M. Larsen N. Overbeek R. Trends Genet. 1997; 13: 497-498Abstract Full Text PDF PubMed Scopus (240) Google Scholar). The PATSCAN output files were analyzed using standard UNIX text processing tools. The total number of occurrences of each class of A-, C-, G-, or U-containing octamers was determined by summing up the number of occurrences of each octamer in that class. The theoretical distribution of each class was determined by a binomial distribution weighted by the fractional proportion of each base in all C. elegans 3′-UTRs. RNAi—C. elegans strains expressing GFP-MEX-5 (JH1448) or GFP-PIE-1 (JH1327) were obtained from the Caenorhabidits Genetics Center and cultured by propagating animals with the roller phenotype. Embryos were harvested from young adult hermaphrodites grown on OP50 food by bleach treatment and then deposited on NGM plates seeded with OP50 or NGM-isopropyl 1-thio-β-d-galactopyranoside plates seeded with mex-3 RNAi food (generously provided by Dr. Craig Mello) and cultured as described (35Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). Embryos were collected from gravid adult hermaphrodites by dissecting the worms in M9 on a 4% agarose pad with a fine gauge needle. DIC and GFP images were collected with live specimens using a Zeiss Axioskop microscope with ×40 or 100 objectives. MEX-5 and MEX-6 Bind to ARE Repeat Elements—To clarify the role of MEX-5 and MEX-6 in development, we set out to test whether these proteins, like TTP, bind with high affinity and specificity to RNA. A recombinant fragment of each protein comprising the TZF domain was purified from bacteria and tested for the ability to interact with an established TTP binding sequence, the AU-rich element (ARE) of TNF-α mRNA (26Brewer B.Y. Malicka J. Blackshear P.J. Wilson G.M. J. Biol. Chem. 2004; 279: 27870-27877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Two approaches were employed to measure affinity to this RNA: electrophoretic mobility shift (EMSA) and fluorescence polarization (FP) assays. Both methods reveal that MEX-5 and MEX-6 bind to TNF-ARE RNA. The EMSA experiments show that multiple binding sites are present in this sequence. The apparent dissociation constant (Kd,app) of MEX-5 for TNF-ARE RNA is 17 ± 1 nm by EMSA and 14 ± 4 nm by FP (Fig. 2, A and B, and Table 1). Similarly, MEX-6 binds to this RNA with an affinity of 4 ± 3 nm by EMSA and 12 ± 3 nm by FP (Fig. 2a). Both proteins are capable of binding to RNA with high affinity. Furthermore, because the EMSA and FP results are nearly equivalent, it is clear that both assays can effectively monitor RNA binding by these proteins.TABLE 1Analysis of MEX-5-TZF binding to RNARNA IDSequenceGene/3′-UTR positionEMSA Kd,appFP Kd,appnmTNF-AREGUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUAGTNF-α, 459–47017 ± 114 ± 4ARE13AUUUAUUUAUUUANAaNA, not applicable55 ± 1597 ± 4TCR1UUGUUUUUUCUCCUUUUCUUUAUAACUUGUglp-1, 242–27229 ± 325 ± 7TCR2UUUCUUUAUAACUUGUUACAAUUUUUGAAAglp-1, 256–28537 ± 1831 ± 9TCR3UUACAAUUUUUGAAAUUCCCUUUUUUGACAglp-1, 270–30066 ± 2252 ± 12TCR4UUCCCUUUUUUGACAGGCUUUUAUUACACUGUAAglp-1, 286–31946 ± 1562 ± 11SubACAAUACUUUUUUAUAUCGGGUCCAACCCGUUUAnos-2, 15–4767 ± 1294 ± 2SubBUACAAGCUUUCACAAACAGAUAGUUUAUnos-2, 53–80>1000400 ± 50SubCCCCGUUCAUAGCCUUUAUUGAUUCCAAAUUUnos-2, 88–11868 ± 1178 ± 13SubDCCCAUCUCACACUUUUCUACGGUAUnos-2, 119–143400 ± 60200 ± 20SubEACCAUUUACUUUUUCUGCUAAUAAUCAAUUAUUAAUAnos-2, 144–18062 ± 1270 ± 12TCR2 U-AAAACAAAAACAAGAAACAAAAAAAGAAANA>1000>1000U30UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUNA29 ± 623 ± 2U15UUUUUUUUUUUUUUUNA150 ± 4140 ± 4C15CCCCCCCCCCCCCCCNA>1000>1000A15GGGGGGGGGGGGGGGNA>1000>1000G15AAAAAAAAAAAAAAANA>1000>1000a NA, not applicable Open table in a new tab TNF-ARE RNA contains several UUAUUUAUU repeat sequences and therefore can bind multiple molecules of TTP (26Brewer B.Y. Malicka J. Blackshear P.J. Wilson G.M. J. Biol. Chem. 2004; 279: 27870-27877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To determine the affinity of MEX-5 for a shorter RNA variant containing only one TTP site, we repeated the binding analyses with ARE13 RNA (AUUUAUUUAUUUA). The apparent dissociation constant for this sequence is 55 ± 15 nm by EMSA and 97 ± 4 nm by FP (Fig. 2). In contrast, TTP binds to ARE13 RNA with 5–10-fold tighter affinity (26Brewer B.Y. Malicka J. Blackshear P.J. Wilson G.M. J. Biol. Chem. 2004; 279: 27870-27877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Together, the results show that MEX-5 binds to UUAUUUAUU repeat sequences, but with an overall affinity that is weaker than TTP. MEX-5 Binds to Regulatory Elements in glp-1 and nos-2 3′-UTRs—Prior work demonstrates that several factors are aberrantly expressed in mex-5 mutants (14Schubert C.M. Lin R. de Vries C.J. Plasterk R.H. Priess J.R. Mol. Cell. 2000; 5: 671-682Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Ectopic expression of five proteins (SKN-1, PIE-1, MEX-1, POS-1, and PAL-1) and reduced levels of two others (GLP-1 and MEX-3) result from mex-5 mutation. Furthermore, MEX-5 is required to activate zif-1, which in turn targets TZF proteins for degradation (16DeRenzo C. Reese K.J. Seydoux G. Nature. 2003; 424: 685-689Crossref PubMed Scopus (136) Google Scholar). Last, recent studies reveal that MEX-5 is required for anterior degradation of mex-1, nos-2, and pos-1 transcripts (21D'Agostino I. Merritt C. Chen P.L. Seydoux G. Subramaniam K. Dev. Biol. 2006; 292: 244-252Crossref PubMed Scopus (60) Google Scholar, 36Tenlen J.R. Schisa J.A. Diede S.J. Page B.D. Genetics. 2006; 174: 1933-1945Crossref PubMed Scopus (12) Google Scholar). Although the results are consistent with MEX-5 regulating a network of maternal genes, this regulation has not been shown to be a direct result of MEX-5 binding to target mRNAs. Extended UAUU sequence repeats similar to the TNF-ARE are not present in the 3′-UTR of any candidate MEX-5 regulatory target. However, several functional regulatory elements have been identified in the 3′-UTR of glp-1 and nos-2 mRNAs (Fig. 3, A and B). Two translational control elements are present in the 3′-UTR of glp-1 mRNA, a spatial control region, and a temporal control region (TCR) (37Evans T.C. Crittenden S.L. Kodoyianni V. Kimble J. Cell. 1994; 77: 183-194Abstract Full Text PDF PubMed Scopus (179) Google Scholar). Furthermore, five elements (subA–E) found in the 3′-UTR of nos-2 mRNA coordinate translational silencing, mRNA localization, and 3′-end formation (21D'Agostino I. Merritt C. Chen P.L. Seydoux G. Subramaniam K. Dev. Biol. 2006; 292: 244-252Crossref PubMed Scopus (60) Google Scholar). To determine whether MEX-5 binds these functional elements, we performed EMSA and FP experiments with 30 overlapping nucleotide fragments of the TCR and all five elements from the nos-2 3′-UTR (Fig. 3, Table 1). Surprisingly, MEX-5 binds with high affinity to all of the TCR fragments and three of the elements from nos-2 mRNA (TCR1–4, subA, subC, and subE, Kd,app ∼ 25–100 nm). MEX-5 binds moderately to nos-2 subD (Kd,app = 200 ± 20 nm) and poorly to nos-2 subB (Kd,app 400 ± 50 nm by FP, >1 μm by EMSA). Because a single shifted species is observed with TCR2 RNA, we decided to investigate the stoichiometry of the complex by repeating the FP experiments with elevated TCR2 RNA concentration (Fig. 3, c and d). The apparent stoichiometry is approximately 1 to 1 (equivalence point N is 0.9 ± 1 by a quadratic fit), demonstrating that there is only one binding site in this RNA and that the recombinant protein is nearly 100% active (Fig. 3d). Consistent with these results, analytical gel filtration chromatography reveals that the TZF domain is predominantly monomeric at concentrations well above the apparent dissociation constant for this RNA sequence (17 μm, Kd,app = 31 ± 9 nm, Fig. 3d). Inspection of the RNA fragments reveals that all of the interacting sequences contain a tract of 6–8 uridines within an 8-nucleotide window. This feature is absent in nos-2 subB, suggesting that MEX-5 requires this element to bind. To test this model, a mutant version of the second TCR fragment where all of the uridines are replaced by adenosine was prepared. As expected, this sequence does not bind to MEX-5 (Table 1). The results show that MEX-5 does not require UAUU repeats to bind to RNA with high affinity, and suggest that MEX-5 can bind to sequences harboring an extended uridine tract. MEX-5 Binds to Polyuridine—TTP displays an 80-fold preference for UAUU repeat sequences over polyuridine (26Brewer B.Y. Malicka J. Blackshear P.J. Wilson G.M. J. Biol. Chem. 2004; 279: 27870-27877Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In contrast, our data show that MEX-5 binds with high affinity to uridine-rich RNAs lacking canonical ARE motifs. To test whether uridine nucleotides are sufficient to promote MEX-5 binding, EMSA and FP experiments were performed with a 30-nucleotide polyuridine sequence. MEX-5 binds to this RNA with an apparent Kd of 29 ± 6 nm by EMSA and 23 ± 2 nm by FP (Fig. 4A). The" @default.
• W2033200059 created "2016-06-24" @default.
• W2033200059 creator A5013480089 @default.
• W2033200059 creator A5015413991 @default.
• W2033200059 creator A5031206696 @default.
• W2033200059 creator A5052146655 @default.
• W2033200059 date "2007-03-01" @default.
• W2033200059 modified "2023-10-18" @default.
• W2033200059 title "Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5" @default.
• W2033200059 cites W1507545655 @default.
• W2033200059 cites W1912833571 @default.
• W2033200059 cites W1944127002 @default.
• W2033200059 cites W1980979311 @default.
• W2033200059 cites W1981809235 @default.
• W2033200059 cites W1985126774 @default.
• W2033200059 cites W1986638937 @default.
• W2033200059 cites W1992525863 @default.
• W2033200059 cites W1993799748 @default.
• W2033200059 cites W2000750967 @default.
• W2033200059 cites W2012160281 @default.
• W2033200059 cites W2020433571 @default.
• W2033200059 cites W2020955446 @default.
• W2033200059 cites W2024177760 @default.
• W2033200059 cites W2026796085 @default.
• W2033200059 cites W2030557396 @default.
• W2033200059 cites W2032363898 @default.
• W2033200059 cites W2039075455 @default.
• W2033200059 cites W2040811742 @default.
• W2033200059 cites W2041701954 @default.
• W2033200059 cites W2045449473 @default.
• W2033200059 cites W2045714098 @default.
• W2033200059 cites W2050054880 @default.
• W2033200059 cites W2051179495 @default.
• W2033200059 cites W2055420305 @default.
• W2033200059 cites W2060809301 @default.
• W2033200059 cites W2061202161 @default.
• W2033200059 cites W2064352437 @default.
• W2033200059 cites W2068054277 @default.
• W2033200059 cites W2083844348 @default.
• W2033200059 cites W2083889014 @default.
• W2033200059 cites W2097581597 @default.
• W2033200059 cites W2098775968 @default.
• W2033200059 cites W2101048878 @default.
• W2033200059 cites W2110940382 @default.
• W2033200059 cites W2111069555 @default.
• W2033200059 cites W2111989690 @default.
• W2033200059 cites W2114056852 @default.
• W2033200059 cites W2118759548 @default.
• W2033200059 cites W2119604062 @default.
• W2033200059 cites W2122103373 @default.
• W2033200059 cites W2122798077 @default.
• W2033200059 cites W2124778982 @default.
• W2033200059 cites W2126036680 @default.
• W2033200059 cites W2137450588 @default.
• W2033200059 cites W2139437132 @default.
• W2033200059 cites W2149871164 @default.
• W2033200059 cites W2151068726 @default.
• W2033200059 cites W2166840544 @default.
• W2033200059 doi "https://doi.org/10.1074/jbc.m700079200" @default.
• W2033200059 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17264081" @default.
• W2033200059 hasPublicationYear "2007" @default.
• W2033200059 type Work @default.
• W2033200059 sameAs 2033200059 @default.
• W2033200059 citedByCount "98" @default.
• W2033200059 countsByYear W20332000592012 @default.
• W2033200059 countsByYear W20332000592013 @default.
• W2033200059 countsByYear W20332000592014 @default.
• W2033200059 countsByYear W20332000592015 @default.
• W2033200059 countsByYear W20332000592016 @default.
• W2033200059 countsByYear W20332000592017 @default.
• W2033200059 countsByYear W20332000592018 @default.
• W2033200059 countsByYear W20332000592019 @default.
• W2033200059 countsByYear W20332000592020 @default.
• W2033200059 countsByYear W20332000592021 @default.
• W2033200059 countsByYear W20332000592022 @default.
• W2033200059 countsByYear W20332000592023 @default.
• W2033200059 crossrefType "journal-article" @default.
• W2033200059 hasAuthorship W2033200059A5013480089 @default.
• W2033200059 hasAuthorship W2033200059A5015413991 @default.
• W2033200059 hasAuthorship W2033200059A5031206696 @default.
• W2033200059 hasAuthorship W2033200059A5052146655 @default.
• W2033200059 hasBestOaLocation W20332000591 @default.
• W2033200059 hasConcept C104317684 @default.
• W2033200059 hasConcept C12426560 @default.
• W2033200059 hasConcept C145103041 @default.
• W2033200059 hasConcept C1491633281 @default.
• W2033200059 hasConcept C2524010 @default.
• W2033200059 hasConcept C2777361361 @default.
• W2033200059 hasConcept C33923547 @default.
• W2033200059 hasConcept C54355233 @default.
• W2033200059 hasConcept C67705224 @default.
• W2033200059 hasConcept C70721500 @default.
• W2033200059 hasConcept C86803240 @default.
• W2033200059 hasConcept C95444343 @default.
• W2033200059 hasConceptScore W2033200059C104317684 @default.
• W2033200059 hasConceptScore W2033200059C12426560 @default.
• W2033200059 hasConceptScore W2033200059C145103041 @default.
• W2033200059 hasConceptScore W2033200059C1491633281 @default.

• next