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Journal of Theoretical Biology 230 (2004) 271–274
RNA mutagenesis and sporadic prion diseases Emmanuel Garciona, Bradley Wallaceb, Laurent Pelletierb, Didier Wionb,* a
INSERM U646, 10 rue Andre Boquel, 49100 Angers, France INSERM U318, CHU Michallon, 38043, Grenoble, France
b
Received 23 February 2004; received in revised form 17 May 2004; accepted 19 May 2004 Available online 17 July 2004
Abstract The extremely low incidence of sporadic prion diseases suggests that they may arise as a rare stochastic event in otherwise healthy animals or humans. Current hypotheses for sporadic prion disease include horizontal transmission, spontaneous conversion of PrpC into PrpSc, and somatic mutation of the Prp gene. Here, we suggest RNA mutation as a possible initial event in the etiology of sporadic prion disease. The proposed model is based on (i) the fact that in Prp-expressing cells, mutations are statistically more likely to occur in the Prp mRNA population than in the corresponding two copies of the Prp gene, and (ii) the absence of RNA repair mechanisms analagous to those found for DNA mismatch correction resulting in a relatively higher rate of RNA mutations. Here, we suggest that translation of mutated Prp mRNA could lead to the synthesis of transient PrpSc which results in the conversion of PrpC into PrpSc and the propagation of a disease-associated isoform. This model points to RNA mutation as a possible mechanism for the generation of sporadic prion diseases and other pathological disorders in which infectious proteins other than PrpSc might be implicated. r 2004 Elsevier Ltd. All rights reserved. Keywords: Prion; RNA; Mutation
1. Introduction In humans, transmissible spongiform encephalopathies (TSE) include Creutzfeld–Jakob disease (CJD), Gerstmann–Straussler-Scheinker . (GSS) disease, kuru, as well as, iatrogenic forms and the new variant of CJD. All these diseases are caused by unusual infectious agents called Prions (Prusiner, 1998). Prion diseases arise either following a mutation in the normal prion (Prp) gene or when an abnormal prion protein molecule (PrPSc) causes a normal prion protein (PrPC) to adopt the conformation of PrPSc (Prusiner, 1998). This conversion of the cellular prion protein into an abnormal isoform results in an amplification of the pathogenic form. It is currently assumed that these diseases have either an endogenous or an exogenous origin. The exogenous origin results from the intake of an infectious PrPSc which then catalyses the conversion of host PrPc into pathogenic PrPSc (kuru, iatrogenic forms and the new variant of CJD). Alternatively, the endogenous origin is usually considered to be the *Corresponding author. E-mail address:
[email protected] (D. Wion). 0022-5193/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtbi.2004.05.014
consequence of a mutation in the Prion protein gene (PRNP), which may be either inherited (familial forms of Creutzfeld–Jacob disease), or spontaneous (somatic mutations corresponding to the sporadic forms of the disease). Sporadic CJD is a rare disorder which occurs at a rate of 0.5–1 per million population per year (McKintosh et al., 2003).
2. RNA mutation and the Prp exception It is generally assumed that the absence of cellular RNA replication combined with the short half-life of the transcripts render the consequences of RNA mutations transitory. In addition, the biological or biochemical consequences of a post-transcriptional mutation in one transcript are expected to be limited as several copies of the corresponding wild-type transcript are present in the same cell. Thus, mRNA mutations should be transient with consequences affecting only a small percentage of the corresponding protein pool. Nevertheless, even if a mutation occurring in a cellular mRNA cannot be physically transmitted by RNA replication, the information corresponding to this mutation can be transferred
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and amplified as a consequence of translation. Interestingly, at least one exception in the transient and limited biological effects of RNA mutations is present if we consider the ‘‘protein only hypothesis’’ of prion replication (Griffith, 1967). In this case, the presence of a mutation in only one Prp mRNA can result in the transitory synthesis of a small amount of PrpSc which acts as a template for the conversion of PrpC into PrpSc and then initiates both the PrpSc replicative cycle and the pathogenic process. Hence in Prp biology, the transient physical nature of a mRNA mutation in the Prp mRNA may persist through the conversion of the translated PrpC into PrpSc and the resulting amplification of PrpSc from the PrpC pool.
3. RNA mutagenesis, RNA mutagens and prion diseases The structural similarity among the four bases forming DNA (A, T, G, C) and RNA (A, U, G, C) suggests that these two molecules may be similarly damaged. However, the biological consequences of most of the RNA mutations are negligible compared to DNA mutations. This may at least partially explain the absence of RNA-repair mechanisms comparable to the sophisticated DNA repair machinery. This point is of special concern as it suggests that lesions will be less efficiently repaired in RNA than in DNA. Like DNA sequences, RNA sequences may be altered spontaneously, chemically or enzymatically.
4. Spontaneous RNA mutation An example of spontaneous base alteration is the spontaneous deamination of cytosine to uracile (Duncan and Miller, 1980). This mutation is immediately recognized and corrected in DNA which, contrary to RNA, does not contain uracile. We presently do not know the rate of deamination of cytosine in mRNA in vivo. However, experiments have demonstrated that the in vitro deamination of cytosine to uracile occurs over 100 times faster in single-stranded than in doublestranded DNA (Frederico et al., 1990). Interestingly, RNA deamination of the internal cytosine contained in proline codons systematically converts them into leucine codons, a substitution of considerable concern in the context of Prp, as proline has the ability to affect the aggregation and function of proteins (Pan et al., 1993; Wood et al., 1995; Reiersen and Rees, 2001). Additionally, RNA cytosine deamination also converts all alanine codons into valine codons. In this regard, it is noteworthy that gene mutations commonly found in hereditary GSS involve these amino acid substitutions (P102L, P105L, and A117 V) (Glatzel and Aguzzi, 2001). Hence, spontaneous cytosine deamination in Prp mRNA
Fig. 1. Sporadic prion disease and the RNA mutation hypothesis: Prp is encoded by a gene located on the short arm of chromosome 20. Several mutations in the coding region of this gene are linked to the inherited forms of CJD and GSS. In sporadic forms of prion disease, mutations of the Prp gene are not found, suggesting that PrpSc may arise by somatic mutation of the Prp gene. However, base damage occurs in both DNA and RNA. For instance, spontaneous base deamination changes cytosine to uracile in both DNA and RNA. This base substitution is efficiently repaired in DNA which does not contain uracile but may persist in RNA. The generation of reactive species following, for example, viral infection or inflammation further increases the frequency of unrepaired Prp RNA mutations and can generate oxidatively damaged RNA bases with unknown coding functions. Errors in RNA editing are an additional source of potential Prp RNA mutations. The transient nature of mRNA and their redundancy has led to the assumption that the effects of RNA mutation are not biologically significant. However, the unique properties of an infectious protein such as PrpSc suggest that the consequences of a RNA mutation may be persistent and amplified through the conversion of PrpC into PrpSc even after the degradation of the altered RNA and of the original PrpSc. The localization of infectious molecules on the cell membrane could be a way to achieve cell to cell propagation.
at codons 102, 105 and 117 could lead to the formation of transient pathogenic PrpSc (Fig. 1). It is important to note that in inherited disease (gene mutation) there is continuous synthesis of mutated proteins over a period of several years whereas in the case of RNA mutation, the synthesis of infectious Prp protein is transient, yet sufficient to initiate a self perpetuating and amplifying process. Furthermore, in the latter case, the amino acid sequence of PrpSc is normal at the time of diagnosis as the mutated mRNA and its corresponding proteins, which are at the origin of the conversion and amplification processes, are no longer present. This difference could be one of the parameters involved in the phenotypic variability of Prp diseases.
5. Chemical RNA mutagenesis Little has been done to assess the biological effects of RNA oxidative damage as the molecule’s transient
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nature suggests that RNA mutations are largely inconsequential. However, RNA is a major site of nucleic acid oxidation and is more susceptible to oxidative damage than DNA (Fiala et al., 1989; Wamer and Wei, 1997; Nunomura et al., 1999; Zhang et al., 1999; Shen et al., 2000). Several reactive species present in the organism can damage both DNA and RNA. For example, the hydroxyl radical OHd modifies both purine and pyrimidine bases (Cadet et al., 1999; Halliwell, 1999), and inflammation-induced NO production can be sufficient to generate additional reactive species able to damage nucleic acids (Nguyen et al., 1992; Shen et al., 2000). Thus, the formation of N2O3 can lead to the deamination of adenine, guanine and cytosine to form hypoxanthine, xanthine and uracil, respectively. On the other hand, ONOO is involved in the nitration or oxidation of guanine (Burney et al., 1999). This suggests that RNAs lack of the type of highly efficient repair mechanisms found in DNA, may result in more unrepaired base alterations in RNA than in DNA secondary to oxidative reactive species or environmental mutagens. This would be true, even if cells have mechanisms for removing oxidized nucleotides from the ribonucleotide triphosphate pool (Mo et al., 1992), for recognizing oxidized RNA molecules (Culbertson, 1999; Hayakawa et al., 2001), and for repairing alkylated RNA (Aas et al., 2003). Consequences of these alterations on the coding sequence are largely unknown, however, reports have been presented suggesting that in DNA 8-Oxoguanine can pair with cytosine and adenine (Maki and Sekiguchi, 1992). This raises the possibility that RNA mutations induced by reactive species could trigger miscoding capable of transiently generating PrpSc (Fig. 1). In this regard it is noteworthy that oxidative stress enhances Prpc protein expression in vitro (Frederikse et al., 2000) , and would favor the synthesis of unglycosylated Prp isoforms (Capellari et al., 1999). In the case of the Prp mRNA, whose half-life has been found to be around 7 h in cultured cells (Pfeifer et al., 1993), such conditions may alter the coding sequence and lead to the transient synthesis of a spectrum of PrpSc thus propagating the conformational change. Interestingly, the fact that a yet uncharacterized single-stranded RNA population distinct from Prp mRNA is required for the in vitro amplification of PrpSc (Deleault et al., 2003) raises the intriguing question of what impact RNA altered bases may have on this process or on non-coding RNA functions (Eddy, 2001).
6. Enzymatic RNA mutagenesis RNA editing is another post-transcriptional process able to change the coding sequence of RNA without affecting DNA. This process includes changes by
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deletion, insertion, as well as A to I, and C to U conversions (Gott and Emeson, 2000). The finding that the primary structure of PrpSc can be the same as that deduced from the Prp gene sequence (Stahl et al., 1993) suggests that constitutive erroneous RNA editing is not necessarily required for the conversion of PrpC into PrpSc. However, this does not exclude the possible involvement of transient aberrant RNA editing as a potential source of PrpSc (Fig. 1).
7. Concluding remarks The possible biological consequences of RNA mutations have been largely ignored due to the transitory nature of RNA, the fact that they are not transmissible, and the assumption that the consequences of a mutation in one mRNA will be counterbalanced by the persistence of several copies of the corresponding wild-type mRNA. However, RNA sequences are more mutation– prone than those of DNA, a situation which may explain the selection of DNA instead of RNA as the hereditary molecule. Interestingly, the biological consequences of RNA mutations have already been described in at least one special process known as ‘‘molecular misreading’’, which leads to frame shift mutations in Alzheimer’s disease patients (Leeuwen et al., 1998). The fact that the frequency at which spontaneous RNA mutations occur can be greatly increased under pathological conditions such as those in which excess reactive species are generated, or in the presence of mutagens, is of special concern. Thus, viral infections (Akaike, 2001), inflammatory reactions (O’Byrne and Dalgleish, 2001), as well as environmental chemicals, may increase RNA mutagenesis, and could lead to the synthesis of infectious PrpSc. An intriguing point to be resolved is that in genetic prion diseases, the mutated protein is synthesized continuously during the entire life, whereas in the proposed models of sporadic prion diseases, smaller amounts of mutated protein can initiate the disease. Two possible explanations are that the existence of compensatory adaptations in genetic CJD may delay the course of the disease, while the presence of host factors in sporadic CJD may stimulate the pathological process. Indeed, the role of such cofactors in the conversion of prion protein has been described in vitro. They include molecular chaperones, RNA molecules, and heparan sulfate (Debburman et al., 1997; Deleault et al., 2003; Ben-Zaken et al, 2003). An alternative, but not exclusive, explanation is that patients with sporadic diseases might have a higher incidence of RNA lesions leading to the synthesis of a heterogeneous population of mutated Prps which could act synergistically during the primary nucleation step to accelerate the initiation of the replicative process. Interestingly, the proposed model can be expanded to
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any RNA miscoding generating a novel transmissible enzymatic or conformational status. This new enzymatic or conformational status does not need to be pathogenic per se, but may act, for example, by changing the substrate specificity. This highlights the importance of considering RNA mutations and agents that increase the RNA mutation rate in the etiology of other rare sporadic idiopathic syndromes.
Acknowledgements We thank Dr. Ravanat for helpful comments.
References Aas, P.A., Otterlei, M., Falnes, P.O., Vagbe, C.B., Skorpen, F., Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E., Krokan, H.E., 2003. Human and bacterial oxidative demethylase repair alkylation damage in both RNA and DNA. Nature 421, 859–862. Akaike, T., 2001. Role of free radical in viral pathogenesis and mutation. Rev. Med. Virol. 11, 87–101. Ben-Zaken, O., Tzaban, S., Tal, Y., Horonchik, .L, Esko, J.D., Vlodavsky, I., Taraboulos, A., 2003. Cellular heparan sulfate participates in the metabolism of prions. J. Biol. Chem. 278, 40041–40049. Burney, S., Caulfield, J.L., Niles, J.C., Wishnok, J.S., Tannenbaum, S.R., 1999. The chemistry of DNA damage from nitric oxide and peroxynitrite. Mutat. Res. 424, 37–49. Cadet, J., Delatour, T., Douki, T., Gasparutto, D., Pouget, J.P., Ravanat, J.L., Sauvaigo, S., 1999. Hydroxyl radicals and DNA base damage. Mutat. Res. 424, 9–21. Capellari, S., Zaidi, S.I.A., Urig, C.B., Perry, G., Smith, M.A., Petersen, R.B., 1999. Prion protein glycosylation is sensitive to redox change. J. Biol. Chem. 274, 34846–34850. Culbertson, M.R., 1999. RNA surveillance: unforseen consequences for gene expression, inhirited genetic disorders and cancer. Trends Genet. 15, 74–80. Debburman, S.K., Raymond, G.J., Caughey, B., Lindquist, S., 1997. Chaperone-supervised conversion of prion protein to its proteaseresistant form. Proc. Natl Acad. Sci. 94, 13938–13943. Deleault, N.R., Lucassen, R.W., Supattapone, S., 2003. RNA molecules stimulate prion protein conversion. Nature 425, 717–720. Duncan, B.K., Miller, J.H., 1980. Mutagenic deamination of cytosine residues in DNA. Nature 287, 560–561. Eddy, S.R., 2001. Non-coding RNA genes and the modern RNA world. Nat. Rev. Genet. 2, 919–929. Fiala, E.S., Conaway, C.C., Mathis, J.E., 1989. Oxidative DNA and RNA damage in the livers of Sprague-Dawley rats treated with the hepatocarcinogen 2-nitropropane. Cancer Res. 49, 5518–5522. Frederico, L.A., Kunkel, T.A., Shaw, B.R., 1990. A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy. Biochemistry 29, 2532–2537. Frederikse, P.H., Zigler, S.J., Farnsworth, P.N., Carper, D.A., 2000. Prion protein expression in mammalian lenses. Curr. Eye Res. 20, 137–143. Glatzel, M., Aguzzi, A., 2001. The shifting biology of prion. Brain Res. Rev. 36, 241–248.
Gott, J.M., Emeson, R.B., 2000. Function and mechanisms of RNA editing. Annu. Rev. Genet. 34, 499–531. Griffith, J.S., 1967. Self replication and scrapie. Nature 215, 1043–1044. Halliwell, B., 1999. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition. Mutat. Res. 443, 37–52. Hayakawa, H., Kuwano, M., Sekiguchi, M., 2001. Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein. Biochemistry 40, 9977–9982. Leeuwen, F.W., Burbach, P.H., Hol, E.M., 1998. Mutations in RNA: a first example of molecular misreading in alzheimer’s disease. Trends Neurosci. 21, 331–335. Maki, H., Sekiguchi, M., 1992. MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature 355, 273–275. McKintosh, E., Tabrizi, S.J., Collinge, J., 2003. Prion diseases. J. Neurovirol. 9, 183–193. Mo, J.Y., Maki, H., Sekiguchi, M., 1992. Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool. Proc. Natl Acad. Sci. 89, 11021–11025. Nguyen, T., Brunson, D., Crespi, C.L., Penman, B.W., Wishnok, J.S., Tannenbaum, S.R.T., 1992. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc. Natl Acad. Sci. 89, 3030–3034. Nunomura, A., Perry, G., Pappolla, M.A., Wade, R., Hirai, K., Chiba, S., Smith, M.A., 1999. RNA oxidation is a pominent feature of vulnerable neurons in Alzheimer’s disease. J. Neurosci. 19, 1959–1964. O’Byrne, K.J., Dalgleish, A.G., 2001. Chronic immune activation and inflammation as the cause of malignancy. Br. J. Cancer 85, 473–483. Pan, K.M., Baldwin, M., Nguyen, J., Gasset, M., Serban, .A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R.J., Cohen, F.E., Prusiner, S.B., 1993. Conversion of a-helices into b-sheets features in the formation of the scrapie prion proteins. Proc. Natl Acad. Sci. USA 90, 10962–10966. Pfeifer, K., Bachmann, M., Schroder, H.C., Forrest, J., Muller, W.E., 1993. Kinetics of expression of prion protein in uninfected and scrapie-infected N2a mouse neuroblastoma cells. Cell Biochem. Funct. 11, 1–11. Prusiner, S.B., 1998. Prions. Proc. Natl Acad. Sci. 95, 13363–13383. Reiersen, H., Rees, R.A., 2001. The hunchback and its neighbours: proline as an environmental modulator. Trends Biochem. Sci. 26, 679–684. Shen, Z., Wu, W., Hazen, S.L., 2000. Activated leukocytes oxidatively damage DNA, RNA and the nucleotide pool through halidedependent formation of hydroxyl radical. Biochemistry 39, 5474–5482. Stahl, N., Baldwin, M.A., Teplow, D.B., Hood, L., Gibson, B.W., Burlingame, A.L., Prusiner, S.B., 1993. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991–2002. Wamer, W.G., Wei, R.R., 1997. In vitro photooxidation of nucleic acids by ultraviolet A radiation. Photochem. Photobiol. 65, 560–563. Wood, S.J., Wetzel, R., Martin, J.D., Hurle, M.R., 1995. Prolines and amyloidogenicity in fragments of Alzheimer’s peptide beta/A4. Biochemistry 34, 724–730. Zhang, J., Perry, G., Smith, M.A., Robertson, D., Olson, S.J., Graham, D.G., Montine, T.J., 1999. Parkinson’s disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons. Am. J. Pathol. 154, 1423–1429.
