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A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein*

  • Rafal A. Bartoszewski
    Affiliations
    Departments of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Michael Jablonsky
    Affiliations
    Departments of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Sylwia Bartoszewska
    Affiliations
    Departments of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Lauren Stevenson
    Affiliations
    Departments of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Qun Dai
    Affiliations
    Departments of Medicine, Hematology, and Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • John Kappes
    Affiliations
    Departments of Medicine, Hematology, and Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • James F. Collawn
    Affiliations
    Departments of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

    The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Zsuzsa Bebok
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd. MCLM 350, Birmingham, AL 35294-0005. Tel.: 205-975-5449
    Affiliations
    Departments of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

    The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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  • Author Footnotes
    * This work was supported, in whole or in part, by Grants R01HL076587 (to Z. B.) and DK060065 (to J. F. C.), CFF, and the Genetically Defined Microbe and Expression Core of the UAB Mucosal HIV and Immunobiology Center Grant R24DK64400 (to J. K.) from the National Institutes of Health.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1, A–C.
Open AccessPublished:July 13, 2010DOI:https://doi.org/10.1074/jbc.M110.154575
      Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.

      Introduction

      Changes in mRNA secondary structures have major biological consequences (
      • Kozak M.
      ,
      • Svoboda P.
      • Di Cara A.
      ,
      • Batey R.T.
      ,
      • Leontis N.B.
      • Lescoute A.
      • Westhof E.
      ). Identification of disease causing mutations that alter the regulatory regions of mRNAs (
      • Cazzola M.
      • Skoda R.C.
      ) and single nucleotide polymorphisms (SNPs) in the coding regions that control gene expression (
      • Kudla G.
      • Murray A.W.
      • Tollervey D.
      • Plotkin J.B.
      ) support this idea. Studies on the dynamics of translation also establish that the secondary structure of the mRNA determines the length of the pause cycles and the rate of translation (
      • Wen J.D.
      • Lancaster L.
      • Hodges C.
      • Zeri A.C.
      • Yoshimura S.H.
      • Noller H.F.
      • Bustamante C.
      • Tinoco I.
      ). Membrane integration and folding of multi-spanning membrane proteins is mainly cotranslational (
      • Alder N.N.
      • Johnson A.E.
      ,
      • Kleizen B.
      • van Vlijmen T.
      • de Jonge H.R.
      • Braakman I.
      ), and therefore mRNA structure-related changes in translational dynamics are likely to influence membrane protein folding.
      Cystic fibrosis (CF)
      The abbreviations used are: CF
      cystic fibrosis
      P-gp
      P-glycoprotein
      ERAD
      endoplasmic reticulum-associated degradation
      CFTR
      cystic fibrosis transmembrane conductance regulator.
      results from mutations in the CFTR gene (
      • Kerem B.
      • Rommens J.M.
      • Buchanan J.A.
      • Markiewicz D.
      • Cox T.K.
      • Chakravarti A.
      • Buchwald M.
      • Tsui L.C.
      ,
      • Riordan J.R.
      • Rommens J.M.
      • Kerem B.
      • Alon N.
      • Rozmahel R.
      • Grzelczak Z.
      • Zielenski J.
      • Lok S.
      • Plavsic N.
      • Chou J.L.
      ,
      • Rommens J.M.
      • Iannuzzi M.C.
      • Kerem B.
      • Drumm M.L.
      • Melmer G.
      • Dean M.
      • Rozmahel R.
      • Cole J.L.
      • Kennedy D.
      • Hidaka N.
      ). CFTR, a multi-spanning membrane glycoprotein in the ABC transporter superfamily, functions as a chloride channel in epithelial cells and regulates complex cellular functions (
      • Collins F.S.
      ,
      • Sheppard D.N.
      • Welsh M.J.
      ). The most common disease-causing mutation in CFTR is the deletion of three nucleotides (CTT) that results in the loss of Phe-508 (ΔF508) and a synonymous SNP (ATT) for Ile-507 (
      • Riordan J.R.
      • Rommens J.M.
      • Kerem B.
      • Alon N.
      • Rozmahel R.
      • Grzelczak Z.
      • Zielenski J.
      • Lok S.
      • Plavsic N.
      • Chou J.L.
      ,
      • Cheng S.H.
      • Gregory R.J.
      • Marshall J.
      • Paul S.
      • Souza D.W.
      • White G.A.
      • O'Riordan C.R.
      • Smith A.E.
      ). Because ΔF508 CFTR is misfolded and subject to rapid endoplasmic reticulum-associated degradation (ERAD) (
      • Ward C.L.
      • Kopito R.R.
      ,
      • Ward C.L.
      • Omura S.
      • Kopito R.R.
      ,
      • Bebök Z.
      • Mazzochi C.
      • King S.A.
      • Hong J.S.
      • Sorscher E.J.
      ), CF has become an excellent model for protein folding disorders (
      • Fadiel A.
      • Eichenbaum K.D.
      • Hamza A.
      • Tan O.
      • Lee H.H.
      • Naftolin F.
      ,
      • Gregersen N.
      ,
      • Cohen F.E.
      • Kelly J.W.
      ). Previous studies, however, have focused on the CFTR protein only, without considering the possible effects of the mutation on CFTR mRNA structure, translation, and cotranslational protein folding.
      The concept that mutations may alter mRNA structure and protein folding led to the experiments presented herein. We compared the structures of WT and ΔF508 CFTR mRNAs via molecular modeling (
      • Zuker M.
      ,
      • Zuker M.
      • Mathews D.H.
      • Turner D.H.
      ) and circular dichroism (CD) spectroscopy (
      • Woody R.W.
      ). To confirm the differences in mRNA structures, we developed an mRNA folding assay based on published methods (
      • Merino E.J.
      • Wilkinson K.A.
      • Coughlan J.L.
      • Weeks K.M.
      ,
      • Wilkinson K.A.
      • Merino E.J.
      • Weeks K.M.
      ,
      • Wilkinson K.A.
      • Merino E.J.
      • Weeks K.M.
      ). We also analyzed the stability and compared the translational rates of WT, ΔF508 (Ile507ATT), and an SNP variant (Ile507ATC) of ΔF508 CFTR mRNAs. Based on the observed differences between the translational rates of two variants of ΔF508 CFTR mRNAs, we developed cell lines and compared the expression of the native (Ile507ATT) and variant (Ile507ATC) ΔF508 CFTR. The results indicate that mRNA “misfolding” may contribute to the severity of the ΔF508 phenotype.

      DISCUSSION

      The experiments presented herein draw attention to the effects of nucleotide deletions and synonymous SNPs on mRNA secondary structure and the consequences of the mRNA structural alterations on translation and protein folding. Our results also demonstrate that high fidelity computational methods (
      • Doshi K.J.
      • Cannone J.J.
      • Cobaugh C.W.
      • Gutell R.R.
      ,
      • Dowell R.D.
      • Eddy S.R.
      ) and biochemical mRNA folding assays (
      • Wilkinson K.A.
      • Merino E.J.
      • Weeks K.M.
      ,
      • Deigan K.E.
      • Li T.W.
      • Mathews D.H.
      • Weeks K.M.
      ) can provide background for studies investigating the dynamics of translation and protein folding.
      Importantly, while several studies proposed that alterations in the secondary structures of mRNAs in the coding region may alter translation and protein folding (
      • Kudla G.
      • Murray A.W.
      • Tollervey D.
      • Plotkin J.B.
      ,
      • Wen J.D.
      • Lancaster L.
      • Hodges C.
      • Zeri A.C.
      • Yoshimura S.H.
      • Noller H.F.
      • Bustamante C.
      • Tinoco I.
      ,
      • Urlinger S.
      • Baron U.
      • Thellmann M.
      • Hasan M.T.
      • Bujard H.
      • Hillen W.
      ,
      • Piazzolla P.
      • Crescenzi A.
      • De Biasi M.
      • Tamburro A.M.
      ), there is limited experimental data in the literature to support this hypothesis. The results presented herein offer significant experimental proof regarding this important prediction.
      In an elegant study, Tinoco and co-workers (
      • Wen J.D.
      • Lancaster L.
      • Hodges C.
      • Zeri A.C.
      • Yoshimura S.H.
      • Noller H.F.
      • Bustamante C.
      • Tinoco I.
      ) emphasized the importance of mRNA secondary structural elements on translational dynamics. Measuring the length of the pauses and translocation steps during translation, they noted that the mRNA hairpin loops influenced the length of pause cycles during translation without affecting translocation between codons. Therefore, these and our results have a direct correlation to what may occur during ΔF08 CFTR biogenesis. The elongated translational pauses during the synthesis of the ΔF508 CFTR NBD1 domain may delay cotranslational folding and signal the quality control (ERAD) machinery. If the elongated pauses initiate, or allow ubiquitination, this could result in early ERAD of ΔF508 CFTR, as demonstrated by previous studies (
      • Zhang F.
      • Kartner N.
      • Lukacs G.L.
      ,
      • Farinha C.M.
      • Amaral M.D.
      ). The observation that ΔF508 CFTR is cotranslationally ubiquitinated and quickly degraded through ERAD (
      • Sato S.
      • Ward C.L.
      • Kopito R.R.
      ) supports this idea. Because strict rules govern the ERAD-associated processes of ubiquitin activation, ubiquitin linkage to proteins and retro-translocation (
      • Vembar S.S.
      • Brodsky J.L.
      ), it is likely that translational rate modifications affect this process. Therefore, it is tempting to speculate that the slower translation of ΔF508 CFTR (Ile507ATT) facilitates ERAD. The results presented in Fig. 4 support this hypothesis because the expression of the variant, Ile507ATC ΔF508 CFTR resulted in higher levels of band B CFTR than the native (Ile507ATT ΔF508 CFTR), likely because it was less accessible to ERAD (Fig. 4B). However, the details how mRNA structural changes affect protein stability remains to be determined.
      Although our studies concentrated on the mRNA secondary structural alterations caused by the deletion of CTT in the human CFTR and introduction of the synonymous codon (ATT) for Ile-507, we also analyzed the codon frequency for Ile in the human genome and in CFTR. We did this based on previous studies suggesting that clusters of rare codons may cause translational pauses (
      • Gupta S.K.
      • Majumdar S.
      • Bhattacharya T.K.
      • Ghosh T.C.
      ,
      • Komar A.A.
      • Lesnik T.
      • Reiss C.
      ,
      • Ramachandiran V.
      • Kramer G.
      • Horowitz P.M.
      • Hardesty B.
      ,
      • Marin M.
      ). We found that that while ATT is a less frequent codon than ATC, ATA is the rarest codon for Ile in the human genome. In CFTR, however, ATT is the most frequently used codon for Ile. Therefore, it is unlikely that the slight reduction of the codon frequency in a single locus (47% (ATC) to 35% (ATT)) with relative synonymous codon usage values changing from 20.9 to 15.8 would significantly affect tRNA availability. A study investigating the role of SNPs on CAT synthesis supports this idea (
      • Ramachandiran V.
      • Kramer G.
      • Horowitz P.M.
      • Hardesty B.
      ). Because excess amounts of Escherichia coli tRNAs did not alter the pause cycles, the authors suggested that mRNA structural alterations, rather than the limited availability of rare tRNAs, were responsible for translational pausing.
      Codon usage, however, has been shown to be important for the folding and function of the MDR1 gene product, P-glycoprotein (P-gp). Studies by Gottesman and co-workers (
      • Kimchi-Sarfaty C.
      • Oh J.M.
      • Kim I.W.
      • Sauna Z.E.
      • Calcagno A.M.
      • Ambudkar S.V.
      • Gottesman M.M.
      ) demonstrated that SNPs that introduced rare codons into MDR1 altered both the structure and ligand specificity of P-gp. Considering that P-gp is an ABC transporter with structural similarities to CFTR (
      • Hoof T.
      • Demmer A.
      • Hadam M.R.
      • Riordan J.R.
      • Tümmler B.
      ), we asked the question whether the mRNA structure, the codon frequency, or the combination of the two were responsible for decreased ΔF508 CFTR translation rates. Our studies on the variant, (Ile507ATC) ΔF508 CFTR, helped to address this question. We showed that replacement of the Ile507ATT with ATC in the mutant resulted in a more WT CFTR-like mRNA structure. Most importantly, both the translational rate and cellular expression levels of the variant (Ile507ATC) ΔF508 CFTR improved compared with the native (Ile507ATT), ΔF508 CFTR. Therefore, these results are consistent with the idea that the reduced translational rates resulted from the mRNA secondary structural differences rather than the rarity of the ATT codon.
      An earlier study provides further support for our hypothesis. It has been proposed that synonymous mutations that alter the cytosine content result in base pairing shifts that have significant effects on mRNA secondary structure (
      • Chamary J.V.
      • Hurst L.D.
      ). Indeed, the ΔF508 mutation in CFTR removes a cytosine from the third position of Ile507 by changing the codon from ATC to ATT. In contrast, if Phe-508 was encoded by the synonym TTC in the WT CFTR, deletion of CTT would not change the cytosine content of the region. Therefore, as suggested by Chamary and Hurst (
      • Chamary J.V.
      • Hurst L.D.
      ) and supported by our results from studies on the Ile507ATC ΔF508 CFTR variant, the mRNA structural instability caused by the removal of cytosine, i.e. a longer single-stranded region in the mutation region, could be responsible for the reduced translational rate.
      Furthermore, the poor correlation between the ΔF508 homozygous genotype and the severity of symptoms implies the involvement of environmental factors and modifier genes in the development of CF (
      • Zielenski J.
      ). Our results presented herein suggest that in addition to CF modifier genes (
      • Knowles M.R.
      ), SNPs that result in mRNA structural changes may also contribute to the differences observed in the severity of symptoms between CF patients homozygous for ΔF508.
      Although we used the CFTR mRNA as our model, genetic mutations are likely to affect the structure of other mRNAs as well. Because, as in a number protein folding diseases involving early degradation of a partially functional protein, the exact amount of functional ΔF508 CFTR sufficient to ameliorate the serious symptoms of CF is not known, it is critical that we understand all of the mutation-associated defects. Therefore, the results presented here provide the foundation for future studies to test the role of mRNA structure on protein folding and ERAD. This type of analysis will improve our understanding of the complex dynamics associated with membrane protein translation, membrane integration and folding.

      Acknowledgments

      We thank Drs. David Bedwell, Kevin Kirk, Casey Morrow, and Lianwu Fu for critical reading of the manuscript and for helpful suggestions.

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