An mRNA Loop/Bulge in the Ferritin Iron-responsive Element Forms in Vivo and Was Detected by Radical Probing with Cu-1,10-phenantholine and Iron Regulatory Protein Footprinting
2002; Elsevier BV; Volume: 277; Issue: 4 Linguagem: Inglês
10.1074/jbc.c100614200
ISSN1083-351X
AutoresYaohuang Ke, Elizabeth C. Theil,
Tópico(s)RNA Research and Splicing
ResumoMessenger RNA (mRNA) regulatory elements often form helices specifically distorted by loops or bulges, which control protein synthesis rates in vitro. Do such three-dimensional RNA structures form in vivo? We now observe formation of the internal loop/bulge (IL/B structure) in the IRE (iron-responsive element) of ferritin mRNA expressed in HeLa cells, using radical cleavage with Cu-phen (Cu-1,10-phenantholine), and protection of the loop/bulge by the regulatory protein (IRP), expressed by cotransfection. Cu-phen, a metal coordination complex (MC) selected because of binding and cleavage at the IL/B in solution, recognized the same site in mRNA in HeLa cells. Endogenous reductants apparently substituted for the sulfhydryl activation of Cu-phen cleavage in solution. Selective RNA IL/B recognition by Cu-phenin vivo is emphasized by resistance to cleavage of a mutated, IL/B IRE in ferritin mRNA. Development of small MCs even more selective than Cu-phen can exploit three-dimensional mRNA or viral RNA structures in vivo to manipulate RNA function. Formation in vivo of the IL/B in the ferritin IRE, which is associated in vitro with greater repression than single IRE structures in other mRNAs, likely contributes to larger derepression of ferritin synthesis in vivo triggered by signals for the IRE/IRP system. Messenger RNA (mRNA) regulatory elements often form helices specifically distorted by loops or bulges, which control protein synthesis rates in vitro. Do such three-dimensional RNA structures form in vivo? We now observe formation of the internal loop/bulge (IL/B structure) in the IRE (iron-responsive element) of ferritin mRNA expressed in HeLa cells, using radical cleavage with Cu-phen (Cu-1,10-phenantholine), and protection of the loop/bulge by the regulatory protein (IRP), expressed by cotransfection. Cu-phen, a metal coordination complex (MC) selected because of binding and cleavage at the IL/B in solution, recognized the same site in mRNA in HeLa cells. Endogenous reductants apparently substituted for the sulfhydryl activation of Cu-phen cleavage in solution. Selective RNA IL/B recognition by Cu-phenin vivo is emphasized by resistance to cleavage of a mutated, IL/B IRE in ferritin mRNA. Development of small MCs even more selective than Cu-phen can exploit three-dimensional mRNA or viral RNA structures in vivo to manipulate RNA function. Formation in vivo of the IL/B in the ferritin IRE, which is associated in vitro with greater repression than single IRE structures in other mRNAs, likely contributes to larger derepression of ferritin synthesis in vivo triggered by signals for the IRE/IRP system. iron-responsive element iron regulatory protein metal coordination complex Cu-1,10-phenantholine internal loop/bulge ferritin Specific messenger RNA (mRNA) structures, identified in solution and exemplified by a set of noncoding mRNA regulatory elements (IRE)1 and proteins (IRP) (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), control rates of protein synthesis (mRNA translation) or mRNA stability. The iso-IRE family, and the two related phosphorylatable regulatory proteins IRP1 and IRP2, are found in mRNAs encoding proteins of iron and oxygen homeostasis and constitute a natural set of combinatorial mRNA/protein interactions that give quantitatively different responses to cellular iron and oxygen signals (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 2Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (101) Google Scholar). Do mRNA tertiary structures such as the ferritin mRNA IRE formin vivo? Metal coordination complexes (MCs), protein nucleases, and alkylating agents have all been used to analyze mRNA solution structure, but MCs are particularly sensitive to the tertiary structure or shape of the RNA binding site (3Murakawa G.J. Chen C.-H. Kuwabara M.D. Nierlich D.P. Sigman D.S. Nucleic Acids Res. 1989; 17: 5361-5375Crossref PubMed Scopus (59) Google Scholar, 4Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (293) Google Scholar, 5Lim A.C. Barton J.K. Biochemistry. 1993; 32: 11029-11034Crossref PubMed Scopus (27) Google Scholar, 6Theil E.C. New J. Chem. 1994; 18: 435-441Google Scholar, 7Muth G.W. Thompson C.M. Hill W.E. Nucleic Acids Res. 1999; 15: 1906-1911Crossref Scopus (17) Google Scholar). MCs have specific geometry and relatively rigid shapes contributed by small organic molecules, coordinated to a metal ion (Fig. 1). If the metal is redox active at physiological conditions, as it is for copper, radical cleavage should occur at the MC/RNA binding site to report on the RNA shape. In solution, Cu-phen binds at RNA loops, bulges, and helix distortions in tRNA, mRNA, and rRNA that are indistinguishable for most alkylating agents and too small for access by protein nucleases (3Murakawa G.J. Chen C.-H. Kuwabara M.D. Nierlich D.P. Sigman D.S. Nucleic Acids Res. 1989; 17: 5361-5375Crossref PubMed Scopus (59) Google Scholar, 4Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (293) Google Scholar, 5Lim A.C. Barton J.K. Biochemistry. 1993; 32: 11029-11034Crossref PubMed Scopus (27) Google Scholar, 6Theil E.C. New J. Chem. 1994; 18: 435-441Google Scholar, 7Muth G.W. Thompson C.M. Hill W.E. Nucleic Acids Res. 1999; 15: 1906-1911Crossref Scopus (17) Google Scholar). We now show that Cu-phen, an MC, detects the tertiary structure in ferritin mRNA in HeLa cells selectively distinguishing the wild type IL/B from a mutant IRE. Such results link the IRE loop/bulge structure observed directly in solution (6Theil E.C. New J. Chem. 1994; 18: 435-441Google Scholar, 8Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (170) Google Scholar, 9Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar, 10Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar) with the predictions from physiological effects of iron on ferritin and other mRNAs that use IRE/IRP regulation (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 2Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (101) Google Scholar) and lay a foundation for studying the behavior of other mRNA structures at the redox conditions in living cells (13Carmel-Harel O. Storz G. Annu. Rev. Microbiol. 2000; 54: 439-461Crossref PubMed Scopus (576) Google Scholar). Plasmid pcDNA3.1-Del-1DV Myc-His (+) (Invitrogen) with sequences deleted between the vector transcription start and HindIII, encoded full-length frog H ferritin mRNA and was derived from 1DV (14Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (58) Google Scholar) using the HindIII site at the 5′ end and theEcoRI site at the 3′ end. pcDNA3.1-Del-1DV/Myc-His (+)-ΔU6 has the same ferritin mRNA insert with deletion of U6 in the IRE, to convert the internal loop/bulge of the Fer-IRE to a C bulge (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar). IRP1 was encoded in pcDNA3.1-Del-IRP1/Myc-HIS (+), which contained the 5′-untranslated region and the coding region of human IRP1 DNA from pGEM-hIRF (from the ATCC) (15Hirling H. Emery-Goodman A. Thompson N. Neupert B. Seiser C. Kuhn L.C. Nucleic Acids Res. 1992; 20: 33-39Crossref PubMed Scopus (47) Google Scholar), amplified by PCR, and inserted into the XhoI andHindIII sites. RNA (1 ng, 30 nucleotides encoding the ferritin IRE regulatory sequence, 5′-32P-labeled, was heated in 40 mm Hepes·Na, pH 7.2, 100 mm KCl to 85 °C for 5 min and slowly cooled to 25 °C after incubation with protein (15 μg of cell protein in the extract in 60 mm KCl, 24 mm Hepes·Na, pH 7.2, 4 mm MgCl2, 5% glycerol) for 30 min at 10 °C, with or without 2% mercaptoethanol (10Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar); the RNA-protein complex formed with each extract is shown. Protein in cell extracts (150 mm) NaCl, 5 mm; EDTA, 10 mm Tris, pH = 7.4, 1% Triton X-100, and protease arrest (Genotechnology) resolved in SDS gels (15 μg of protein/lane), were transferred to nitrocellulose and detected with c-Myc antibodies (Invitrogen) and horseradish peroxidase-IgG (Pierce). HeLa cells (8 × 104 cells/well) cultured in Dulbecco's modified Eagle's medium + 10% fetal bovine serum for 16–18 h at 37 °C, 5% CO2 were incubated with plasmid DNA (1 μg/well), PLUS (Invitrogen) reagent (6 μl PLUS/well, and 4 μl of LipofectAMINETM/well) (Invitrogen) LipofectAMINETM, in six-well plates. To express high levels of IRP1 for "footprinting" on the IRE, HeLa cells were cotransfected with plasmids encoding ferritin and IRP1 as described for RNA expression, except the cells (1 × 106 cells/plate) were plated in a 10-cm plate the day before transfection and cotransfected with 1.5 μg of pcDNA3.1-Del-1DV/Myc-His (+) ferritin mRNA and 5.0 μg of pcDNA3.1-Del-IRP1/Myc-His(+) or with pcDNA3.1/Myc-His/LacZ plasmid DNA encoding β-galactosidase in the LacZ sequence, as a control for cotransfection (cotransfection frequency (β-galactosidase) ∼20%). 48 h after transfection and 1 h of culture in prewarmed Opti-MEM (Invitrogen), Cu-phen was added (to 225 μm) for 30 min. Cells were rinsed with phosphate-buffered saline, collected by scraping, and washed one time with phosphate-buffered saline; cell pellets were frozen at −80 °C. Cell survival (trypan blue exclusion), ∼60% with Cu-phen, was likely diminished because of cutting tRNA. (Survivals should increase in the future with identification and use of more specific MCs.) Total RNA, isolated with an RNeasy Mini kit (Qiagen) and precipitated with ethanol, was the template for 32P-labeled cDNA synthesized with a labeled primer, bound 40 nucleotides downstream from the ferritin IRE. Mixtures of cDNA were resolved on calibrated urea-acrylamide gels as described previously (16Harrell C.M. McKenzie A.R. Patino M.M. Walden W.E. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4166-4170Crossref PubMed Scopus (77) Google Scholar). Sites of Cu-phen binding/cleavage were identified by the increase in shorter cDNA fragments (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar) and quantitated by a PhosphoImager (Molecular Dynamics) with ImageQuant software. RNA dissolved in water, and stored at −80 °C until use, was heated at 65 °C for 3 min, quickly cooled to 25 °C, and reacted in 25 mm Hepes·Na, pH 7.2, 40 mm KCl, 18 μm Cu-phen, and 0.05% mercaptopropionic acid (to reduce copper); after 5 min at 25 °C, 2,9-dimethyl-1,10-phenanthroline was added to 7 mm as a "quench," with the same cDNA analysis used with RNA isolated from HeLa cells. Cu-phen sites in the IRE of ferritin mRNA in HeLa cells are the same as an IRE 30-mer or in poly(A)+ RNA folded in solution. Deletion of a single nucleotide, U6 (see Figs. 1 and 2E), changed the IRE structure in ferritin mRNA in solution and prevented Cu-phen binding (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar). The ferritin IRE (Fer-IRE) structure was selected for the study of mRNA structure in vivo (in cultured HeLa cells), because the IL/B of the Fer-IRE contributes to selectivity of regulatory protein binding in solution (10Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In addition, Cu-phen binds selectively in solution, discriminating between the IL/B and a C-bulge (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar). High IRE structural specificity in solution is also indicated by the specific MC site that is blocked by magnesium and is conformationally sensitive to protons (9Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar). Cu-phen has the added advantage of being relatively hydrophobic and should cross cell membranes. HeLa cells were chosen for the study because they display the IRE-mRNA regulation typical of many cell types (17Cairo G. Pietrangelo A. Biochem. J. 2000; 352: 241-250Crossref PubMed Scopus (276) Google Scholar). When Cu-phen was added to cultures of HeLa cells in which ferritin mRNA was expressed at high levels from a transfected plasmid in HeLa cells, the IRE was recognized and cleaved by Cu-phen (Figs. 1B and 2, Aand C). Cu-phen cut the RNA at the IL/B, showing that the mRNA folded in vivo with the same specific IL/B structure of the Fer-IRE observed in solution by NMR spectroscopy and by nuclease probing (9Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar). In addition, the results show that the Cu-phen was able to enter cells and recognize specific RNA sites. IRE context in the mRNA was important for IL/B formation in HeLa cells, since the IRE inserted upstream from β-galactosidase sequences did not form the IL/B in vivo. Cleavage of RNA in HeLa cells was the same whether or not exogenous reductants were added, indicating the availability of endogenous reductants (13Carmel-Harel O. Storz G. Annu. Rev. Microbiol. 2000; 54: 439-461Crossref PubMed Scopus (576) Google Scholar). In solution, Cu-phen cleavage of RNA requires the addition of a reductant (4Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (293) Google Scholar). Sites of Cu-phen binding (mRNA cleavage) in the IRE were determined by reverse transcriptase run-off using cleaved mRNA recovered from the treated cells as the template, and a primer binding near the IRE (Fig. 2). cDNA fragments were separated in DNA sequencing gels calibrated with cDNAs synthesized from intact mRNA and mixtures of deoxy- and dideoxynucleotide triphosphates (see Refs. 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar and 12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar as examples). The background reaction, due to reverse transcriptase pausing, is ∼4 times lower than reverse transcriptase run-off from RNA fragments produced by Cu-phen cleavage. Several sites outside the IL/B of the ferritin IRE were Cu-phen binding sites in HeLa cells, but not in the in vitro transcripts of the ferritin mRNA, such as residue −13 (Fig. 2C) at the end of the IRE-flanking region helix. Cu-phen recognizes the −13 site not only in ferritin mRNA transcripts in HeLa cells, but also in natural ferritin mRNA (poly(A)+) that was synthesized in frog reticulocytes of the embryonic (tadpole) lineage, but analyzed in vitro with specific primers (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 16Harrell C.M. McKenzie A.R. Patino M.M. Walden W.E. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4166-4170Crossref PubMed Scopus (77) Google Scholar). Such mRNAs likely differ from in vitrotranscripts in the structure of the cap, which suggests that the cap structure influences the conformation of the ferritin IRE flanking region. Previous structure/function links observed between the mRNA cap and the ferritin IRE are conserved length, conserved sequences, and regulatory effects of altering the distance between the cap or mutating conserved bases flanking the IRE (11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 14Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (58) Google Scholar, 18Goossen B. Hentze M.W. Mol. Cell. Biol. 1992; 12: 1959-1966Crossref PubMed Scopus (111) Google Scholar). Selectivity of Cu-phen for the IL/B structure of the Fer-IRE in vivo was illustrated by the resistance of mutant ferritin mRNA, Fer-IRE Δ U6, to Cu-phen cleavage. Deletion of a single IL/B nucleotide (Fer-IRE Δ U6, Fig. 2E), converted the IL/B to a C-bulge found in other mRNAs with isoIREs, such asm-aconitase and the transferrin receptor (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Resistance of Fer-IRE Δ U6 mRNA to Cu-phen cleavage in HeLa cells (Fig. 2,B and D) illustrates the specificity of IRE folding in vivo as well as structural selectivity of Cu-phenin vivo. IRP1, an IRE-binding protein expressed from a cotransfected plasmid, protected the IL/B (G26, G27) in ferritin mRNA from Cu-phen cleavage in vivo (HeLa cells) (Fig.3). IRP1, expressed in HeLa cells, was characterized in cell extracts by immunoreactivity and RNA binding of an IRE (Fig. 3B). IRP1 was selected because of greater stability in vivo (19Guo B. Brown F.M. Phillips J.D. Yu Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Samaniego F. Chin J. Iwai K. Rouault T.A. Klausner R.D. Biol. Chem. 1994; 269: 30904-30910Abstract Full Text PDF Google Scholar). In cells cotransfected with plasmids encoding both ferritin mRNA and IRP1, Cu-Phen activity at the IL/B decreased to the background level of G7 (Fig. 3C). An earlier study using permeabilized cells, and a large protein nuclease (RNase T1) (21Bertrand E. Fromont-Racine M. Pictet R. Grange T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3496-3500Crossref PubMed Scopus (39) Google Scholar) was limited to demonstrating IRP protection of a protruding residue, G16, in the hairpin loop at the tip of the IRE terminal loop and could not, in contrast to Cu-phen, report on tertiary structures such as the internal loop/bulge within the middle of the IRE helix. When β-galactosidase protein was expressed, rather than the IRE-binding protein IRP1, no protection of the IL/B occurred, and G26 and G27 were cleaved by Cu-phen added to the cells (Fig.3D). Such results demonstrate the specificity of IRP binding and the IRP "footprint" over the IL/B in vivo. Observation of the mRNA-specific IL/B in ferritin IRE in vivo (Figs. 2 and 3), using the MC reporter Cu-phen, confirms the tertiary structure of the mRNA element observed in solution (9Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar,12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar). Specificity of IRE structure in each mRNA is the basis for the natural combinatorial mRNA regulation displayed by the IRE/IRP system (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In the future, combinatorial chemical synthesis can improve selectivity and recognition of control structures such as the IRE (1Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), AU-rich elements (22Chen C.U. Shy A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar), and internal ribosome entry sites (23Holcik M. Sonenberg N. Korneluk R.G. Trends Genet. 2000; 16: 469-473Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) so that RNA structure may be further studied and possibly manipulatedin vivo. (Note that since Cu-phen is an "off-the-shelf" reagent, despite its IRE-IL/B selectivity, Cu-phen also cleaves tRNA (4Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (293) Google Scholar), and a site at the base of the BIV-tar element, inserted into β-galactosidase mRNA in HeLa cells.) 2E. Theil, unpublished observations. Targeting three-dimensional mRNA structure in vivocomplements the manipulations of three-dimensional protein structure in cells, a common strategy for understanding protein function or for developing medical therapies. RNA targeting shares high cell specificity with proteins, but has the added advantage of lower copy number and target size. Understanding RNA function in vivo is only one potential use of MCs targeted to specific RNAs. Inactivation of viral mRNAs or prophylactic ferritin synthesis for protection from iron toxicity are other possible uses of MCs. The selectivity of targeting IRE structure with small molecules is matched by the natural selectivity of IRP/IRE interactions in different mRNAs. For example, the iron induction of proteins encoded in different IRE-mRNAs had quantitatively different responses to iron in the same tissue of whole animals (2Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (101) Google Scholar, 24Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Induction of ferritin synthesis, for example, was much greater than m-aconitase synthesis. (Effects of iron on m-aconitase regulation were small enough to have gone unnoticed until the IRE sequence was detected, whereas mRNA regulation of ferritin had been known long before the RNA sequence was determined (25Shull G.E. Theil E.C. J. Biol. Chem. 1982; 257: 14187-14197Abstract Full Text PDF PubMed Google Scholar, 26Shull G.E. Theil E.C. J. Biol. Chem. 1983; 258: 7921-7923Abstract Full Text PDF PubMed Google Scholar).) The differences between the iron-responses of ferritin and m-aconitase mRNA in vivowere attributed in part to the behavior of the IL/B in the ferritin IRE in solution and cell-free protein synthesis (10Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar). Linking the solution studies on IRE structures (e.g. Refs. 8Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (170) Google Scholar, 9Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar, 10Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Ke Y. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 12Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar,14Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (58) Google Scholar, 15Hirling H. Emery-Goodman A. Thompson N. Neupert B. Seiser C. Kuhn L.C. Nucleic Acids Res. 1992; 20: 33-39Crossref PubMed Scopus (47) Google Scholar, 16Harrell C.M. McKenzie A.R. Patino M.M. Walden W.E. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4166-4170Crossref PubMed Scopus (77) Google Scholar) and physiological regulation of proteins encoded in IRE-mRNAs by the observation of the IL/B structure in vivo (Figs. 2 and 3) emphasizes the importance of tertiary structure in mRNA regulatory elements.
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