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Molecular Psychiatry (2005) 10, 69–78 & 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00 www.nature.com/mp FEATURE REVIEW Expanding the ‘central dogma’: the regulatory role of nonprotein coding genes and implications for the genetic liability to schizophrenia DO Perkins1, C Jeffries2 and P Sullivan3 1 Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Genetic Control Research, Research Triangle Park, NC, USA; 3Departments of Genetics, Psychiatry, and Epidemiology, Chapel Hill, NC, USA It is now evident that nonprotein coding RNA (ncRNA) plays a critical role in regulating the timing and rate of protein translation. The potential importance of ncRNAs is suggested by the observation that the complexity of an organism is poorly correlated with its number of protein coding genes, yet highly correlated with its number of ncRNA genes, and that in the human genome only a small fraction (2–3%) of genetic transcripts are actually translated into proteins. In this review, we discuss several examples of known RNA mechanisms for the regulation of protein synthesis. We then discuss the possibility that ncRNA regulation of schizophrenia risk genes may underlie the diverse findings of genetic linkage studies including that proteinaltering gene polymorphisms are not generally found in schizophrenia. Thus, inadequate or mistimed expression of a functional protein may occur either due to mutation or other dysfunction of the DNA coding base pair sequence, leading to a dysfunctional protein, or due to post-transcriptional events such as abnormal ncRNA regulation of a normal gene. One or more ‘schizophrenia disease genes’ may turn out to include abnormal transcriptional units that code for RNA regulators of protein coding gene expression or to be proximal to such units, rather than to be abnormalities in the protein coding gene itself. Understanding the genetics of schizophrenia and other complex neuropsychiatric disorders might very well include consideration of RNA and epigenetic regulation of protein expression in addition to polymorphisms of the protein coding gene. Molecular Psychiatry (2005) 10, 69–78. doi:10.1038/sj.mp.4001577 Published online 21 September 2004 Keywords: miRNA; ncRNA; schizophrenia; epigenetics; BDNF; DISC1; DRD2; RELN Noncoding RNA, mRNA stability, and protein coding gene regulation The ‘central dogma’ of genetics is that DNA is transcribed into messenger RNA (mRNA), and mRNA in turn is translated into proteins. The central dogma is certainly valid in that it defines what proteins are possible to make, but in recent years it has become evident that much more genetic information exists in the genome, namely the information used in controlling the timing and rates of the protein manufacturing processes. In higher organisms only a minority of genetic transcripts (2–3%) code for proteins.1,2 Nonprotein coding RNA (ncRNA) refers to mRNA that is transcribed from DNA but not translated into protein. Rather than being ‘junk’ DNA (ie an evolutionary relic) some nonprotein coding transcripts may in fact play a critical role in regulating gene expression and so organizing the development and maintenance of complex life. For example, a study Correspondence: DO Perkins, MD, MPH, Department of Psychiatry, CB 7160, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7961, USA. E-mail: [email protected] Received 12 April 2004; revised 18 June 2004; accepted 26 July 2004 identified the highly conserved regions across 12 vertebrate species in a 12 mb orthologous genetic region and found most (80%) of the highly conserved regions to be located in introns or in intergentic regions, suggesting that these nonprotein coding regions play an essential, but as yet largely unknown regulatory role.4 In addition, the potential importance of ncRNAs is suggested by the observation that the complexity of an organism is poorly correlated with its number of protein coding genes, but highly correlated with its number of ncRNA genes.3 The rate and amount of protein synthesized is related to the quantity of mRNA available, which in turn is a function of the rate of DNA transcription, the rate of mRNA degradation, and other factors. Both transcription and mRNA degradation appear to be tightly regulated, with ncRNAs potentially involved in the developmental timing and quantity of protein synthesis.4 For example, ncRNA may regulate protein synthesis by decelerating or accelerating mRNA degradation.5–8 Control mechanisms may target the mRNA degradation enzymes or some other aspect of the propensity of the mRNA to be degraded, thus impacting the steady concentration (ie ‘half-life’) of the mRNA.9 These newly discovered control mechan- Expanding the ‘central dogma’ DO Perkins et al 70 isms for protein expression are often referred to as the ‘tip of the iceberg’, anticipating that yet undiscovered regulatory functions of ncRNAs are likely to be critical to the development and function of complex organisms.2 In classical Mendelian disorders, mutations generally lead to striking alterations in protein structure that impair function, resulting in disease. This theoretical perspective has led investigators to emphasize investigation of nonsynonymous base pair (bp) changes as candidate disease genes.10 However, recent genome scans for complex diseases have found that for the genomic location where statistical evidence is strongest, the relevant markers are not generally located in exons and so cannot directly cause pathological change in the protein product. An example includes the association of noninsulindependent diabetes mellitus with intronic genetic variation in calpain 10 gene (CAPN10).11 Relevant to this paper are the replicated findings of association of schizophrenia with a specific noncoding singlenucleotide polymorphism (SNP) haplotype in the gene for dysbindin (DTNBP1)12 and neuregulin 1 (NRG1).13 Furthermore, there is evidence that nonsynonymous polymorphisms may alter the rate of gene expression in humans through as yet unknown mechanisms.14 It is thus becoming apparent that altered regulatory control of the transcription or the translation of a gene may contribute to disease risk. Below are described several noteworthy examples of mRNA regulatory mechanisms that may play a role in complex diseases. Several speculative examples are then provided to illustrate how impairment of mRNA regulation could potentially play a role in schizophrenia. Table 1 Micro-RNA (miRNA) Small RNA segments form a critical cellular regulatory function, directly affecting the timing and rate of protein expression (see Table 1). Science Magazine highlighted the potential importance of these small RNAs to understanding the evolution and operation of the genome, naming this discovery the ‘Breakthrough of the Year’.7,15 A newly described ‘family’ of small ncRNA segments is miRNA. There are an estimated 200–255 human miRNA genes, constituting about 1% of predicted human genes.16,17 Demonstrated function of animal miRNAs includes the regulation of posttranscriptional processing of mRNA in neuronal and general development, apoptosis, cell differentiation, and cell proliferation.18,19 The precursor of a miRNA is initially transcribed (from DNA) to a single strand of RNA, which then folds over forming a double-stranded (ds) ‘hairpin’ precursor. The precursor miRNA contains a short (B17–24) nucleotide ‘mature’ sequence imbedded in the longer (B50–120) nucleotide sequence.18,16 The mature sequence itself is highly conserved across species, in contrast to the rest of the precursor miRNA sequence, which shows evolutionary divergence.16 The precursor miRNA is exported from the nucleus to the cytoplasm of a cell, where it is then processed by the enzyme DICER into a dsRNA (from the stem of the hairpin) that includes the mature sequence and its partially homologous complement. This dsRNA is further processed so that the mature sequence becomes part of the RNA-induced silencing complex (RISC). In animals, the nucleotides of the mature miRNA sequence are imperfectly complementary to a bp sequence of the target mRNA 30 -untranslated region (UTR). Other types of small, ncRNA, such as Definition of some recently described ncRNAs Acronym Name Definition ncRNA Noncoding RNA dsRNA Double-stranded RNA miRNA Micro-RNA siRNA Small interfering RNA RNAi RNA interference snRNA Small nuclear RNA Small nucleolar RNA A group of RNAs that are transcribed but are not translated into a protein, some fraction with regulatory functions. Single-stranded RNA is unstable and quickly degrades unless associated with a protein complex. Single-stranded transcribed RNA may fold over forming a double-stranded ‘hairpin’ that is protected from degrading enzymes. Single strand of B17–25 nucleotides with regulatory functions when associated with a protein complex. In animals, miRNAs inhibit translation by binding with imperfect homology to the untranslated region of mRNA, inhibiting translation of mRNA into protein. Single strand of B21–23 nucleotides with regulatory functions when associated with a protein complex. siRNAs bind to mRNA with perfect bp homology causing mRNA degradation. Synthesized double-stranded RNA introduced into the cell may be processed in a manner similar to endogenous siRNA and used to ‘silence’ a gene of interest. The mechanism where double-stranded RNA is processed to a single strand that targets mRNA resulting in mRNA degradation or translational inhibition. Reside within the nucleus of eukaryotic cells and involved in RNA splicing or histone mRNA termination. Participate in methylation or ribosomal RNA modification or processing. snoRNA Molecular Psychiatry Expanding the ‘central dogma’ DO Perkins et al ‘short interfering RNA’ (siRNA), as well as plant miRNA are perfectly complementary to the target mRNA. While ncRNA–target interactions with perfect complementarity tend to result in mRNA cleavage and degradation, ncRNA–target interactions with imperfect complementarity tend to result in mRNA translational inhibition. Since in animals, miRNA– target interactions typically involve imperfect bp complementarity, miRNAs act to regulate gene expression by binding to target mRNAs, thereby block- ing ribosome processing and inhibiting mRNA translation (see Figure 1). The first description of miRNA:target interaction and of miRNA function was in the nematode, Caenorhabditis elegans.16 Two proteins important in C. elegans development are lin-4, which is expressed in the early larval stage, and lin-14, expressed in the later larval stage. In a series of insightful studies, it was discovered that a region of the lin-4 gene codes for a miRNA (cel-lin-4) whose mature region binds to 71 Figure 1 ncRNA and mRNA interactions. mRNA transcribed from a protein coding gene (1) may be translated into a protein (2). siRNA and miRNA interact with RISC (3). RISC ncRNA complex may then interact with the transcribed mRNA (4). Perfectly complementary ncRNA and mRNA binding will then subject the mRNA to cleavage and degradation (5). Imperfectly complementary ncRNA:mRNA may allow the mRNA to bind with ribosomes, but interferes with translation (6). Either mechanism precludes protein expression (7). Molecular Psychiatry Expanding the ‘central dogma’ DO Perkins et al 72 the 30 -UTR of the mRNA from the lin-14 gene. Since the 30 -UTR is needed for correct translation of mRNA to protein, the binding of the mature region of the miRNA to the target 30 -UTR prevented translation of the lin-14 mRNA.20 The investigators demonstrated that loss of the cel-lin-4 miRNA results in the overexpression of lin-14 at a developmentally inappropriate time. Thus, an apparent function of cel-lin-4 miRNA is to regulate the lin-14 gene translation at specific developmental time points. Since this initial discovery, there have been numerous descriptions of miRNA mature sequences that are partially complementary to the 30 -UTR of a protein coding gene.21,22 A regulatory role of miRNA affecting mRNA degradation and stability (in plants) and translation (in animals) is now known.16,18 In addition to interfering with translation of mRNA, miRNA may conversely increase gene expression by binding with some other regulatory RNA, in effect inhibiting inhibition.23 had very low levels of Makorin1 protein, Makorin1 mRNA and Makorin1p mRNA, resulting in the lethal phenotype. Another experiment demonstrated that Makorin1 mRNA has a much greater half-life in the presence of Makorin1p as compared to the half-life in the absence of Makorin1p. Thus, the pseudogene somehow reduces the degradation rate of the gene mRNA. The presence of the intact pseudogene is needed for correct expression rate of the Makorin1 gene. Potential explanations for Makorin1p regulation of Makorin1 expression include that the Makorin1p mRNA could interfere with the degradation of the Makorin1 mRNA by acting as a ‘decoy’ for enzymes degrading Makorin1 mRNA, thus increasing the amount of mRNA available for translation into protein.27 A second possible explanation is that the pseudogene may act as a decoy for transcriptional suppressor proteins, an instance of inhibiting inhibition of Makorin1 gene transcription to mRNA.28 Functional pseudogenes A pseudogene has a similar bp sequence to a protein coding gene, but is missing critical bp sequences required for translation. A pseudogene may be transcribed, but the resultant mRNA is not translated into a protein. Numerous (B20 000) pseudogenes have been identified in the human genome, and many genes have multiple pseudogenes, often located on different chromosomes than the gene itself.24,25 Until very recently, pseudogenes were considered nonfunctioning genetic ‘mistakes’ or ‘fossilized’ remnants of discarded genes. Evidence for a possible biological role for at least some pseudogenes comes from the observation that many pseudogenes appear to be conserved across species and thus do not show the pattern of gradual, evolutionary sequence degradation as would be expected for a nonfunctioning genetic region.25 Pseudogenes that reside near their gene are thought to contribute to gene mutation through the process of gene conversion. Gene conversion occurs during meiotic crossover events, where there is unequal exchange of genetic material between a gene and near-homologous pseudogene that is in close proximity to the gene. Gene conversion is a proposed mechanism of PRODH gene mutation, implicated in the etiology of schizophrenia.26 Direct evidence of pseudogene function has come from the discovery of a functional pseudogene in mice. The pseudogene was discovered fortuitously while conducting a study which involved inserting a Drosophila gene Sex-Lethal randomly into the mouse genome.27 This gene insertion was fatal for one line of mice. Surprisingly, the Sex-Lethal gene had been inserted in the middle of a pseudogene, Makorin1p, located on murine chromosome 5. This pseudogene is located on a different chromosome than that of the functional gene, Makorin1, located on chromosome 6. Further study demonstrated that the Makorin1p was normally transcribed during fetal and postnatal development. Mice with the disrupted pseudogene mRNA stability The rate of mRNA degradation by various enzyme systems impacts the rate of protein synthesis, since an individual mRNA may be used in translation multiple times, depending on how long the mRNA remains intact in the cytosol. The steady-state concentration of mRNA is thought to be tightly regulated, although the mechanisms controlling mRNA concentration stability are not yet understood.9,29 The tertiary structure of mRNA may impact its rate of degradation and translation. Some amino acids can be specified in the translation process by multiple RNA codons (eg CCU, CCC, CCA, and CCG all code for proline). Thus, variations of DNA base sequences among gene polymorphisms often do not impact the translated amino-acid sequence and thus the synthesized protein. Such sets of codons coding for one amino acid are termed ‘synonymous’ genetic polymorphisms. However, while the alternative codons do not influence primary or secondary protein structure, they may not be silent. Rather, alternative codons may affect the tertiary structure of mRNA, thereby affecting mRNA degradation rate, mRNA half-life, and hence the rate of protein expression.30 Molecular Psychiatry Epigenetic factors Epigenetic factors include a layer of genetic information and control embodied in the chromatin—the proteins and other chemicals that surround a chromosome. The chromatin serves to isolate and protect DNA from degradation, transcription, and interactions with other proteins including ncRNAs. Epigenetic information is remodeled in response to other genetic events and environmental signals. The chromatin partitions active and silent chromosomal regions, and the partition boundaries change in development and also in response to environmental factors.31 If genetic expression is altered through epigenetic mechanisms by an environmental insult, it may remain altered for the life of the individual, Expanding the ‘central dogma’ DO Perkins et al potentially causing disease by misexpression of a protein.32 Chromatin boundary mechanics may be either mutually reinforcing or mutually inhibitory. The result may be polarization of chromatin domains, so that they are fixed in a state of transcriptional activity or silence.33 One prominent epigenetic mechanism is methylation, meaning the inactivation of a gene by attachment of methyl radicals to instances of C (cytosine) with following G (guanine). Epigenetic proteins may control genetic expression in a single locus, a chromosomal region, or an entire chromosome.31 The epigenetic effect of ‘imprinting’ allows the activation of only one allele from the copies in maternal and paternal chromosomes. Thus, inheriting a chromatin structure that consistently chooses for suppression of the same parental locus of a trait may result in a parent-of-origins effect where a trait can be inherited only from one parent. Inheriting this chromatin structure means propagation of gene activity partitions from one cell generation to another (mitosis) or from one individual to progeny (including meiosis). Environmental factors early in life can modify chromatin structure and thereby stably alter the expression of genes into adulthood, that is, the genome reacts to its experiences.33 Also, identical twins exposed to different environmental factors can differentially express disease due to the presence or absence of correct imprinting.34 Hence, epigenetic effects may eventually help explain the complex roles of environment and inheritance in the etiology of schizophrenia. RNA mechanisms may be involved in epigenetic gene regulation. Small (short), interfering RNAs, siRNAs, might be involved in chromatin silencing or DNA methylation, and they might also have roles in the regulation of both conventional genes (that code for proteins) and RNA-only transcriptional units.31 The genetics of schizophrenia Schizophrenia is clearly a heritable disorder with overwhelming evidence that genetic factors play an important role.35,36 However, the specific genetic factors that contribute to schizophrenia are not known, despite decades of research. The basic strategies used to investigate the genetic basis of schizophrenia include family linkage studies and candidate gene association studies. A recent metaanalysis of linkage studies defined 19 areas on 15 chromosomes where there is evidence across studies for involvement in the etiology of schizophrenia (only one area, 2p12-q22, met statistical significance when adjusting for multiple comparisons).37 The areas defined by these 19 linkage regions consists of about 565 million bp’s, representing approximately 18% of the 3 billion bp’s that make up the human genome. Additional chromosomal regions have also been associated with schizophrenia risk in other linkage studies.37,38 Converging findings from neurobiological studies implicate abnormal function of prefrontal cortical and limbic area circuits that involve dopamine, glutamate, and GABA in the pathology of schizophrenia.39–42 In addition, there is strong evidence that altered brain development is involved in schizophrenia risk.42 Recent linkage studies using closely spaced SNPs have further narrowed possible linkage areas and then, based on SNP association, identified candidate schizophrenia risk genes. The risk genes identified notably include genes that are involved in the metabolic pathways of these neurotransmitter or implicated receptors, or in neurodevelopment. This strategy has identified several schizophrenia risk genes, with varying levels of genetic, molecular biological, and theoretical evidence.43,44 In particular, two leading schizophrenia risk genes, NRG1 and DTNBP1, have disease-associated polymorphisms that do not result in the expression of an abnormal protein in the manner generally found for classical Mendelian disorders.45,12 73 A third explanation One hypothesized explanation for the diverse genetic associations found for schizophrenia is a ‘polygene model,’ where each gene locus has a small effect so that schizophrenia arises as the aggregate consequence some mixture of multiple risk genes or environmental factors. A second hypothesized explanation is a ‘locus heterogeneity model’, where the risk is large, but only a small proportion of individuals are vulnerable. A third explanation for the diverse findings of genetic linkage studies and the conflicting evidence supporting a particular gene polymorphism is that regulation of schizophrenia risk gene(s) expression is disrupted. Dysregulation by ncRNA of the rate of transcription or of translation of a normal gene may lead to abnormal gene expression that is phenotypically similar to disruption of the gene itself. Thus, inadequate or mistimed expression of a functional protein may occur either due to disruption of the DNA coding bp sequence, leading to a dysfunctional protein, or due to abnormal ncRNA regulation of a normal gene. ncRNA regulation of gene transcription and translation may be particularly important during brain development,19 and so is consistent with a neurodevelopmental diathesis for schizophrenia. ncRNA may also regulate gene expression in adulthood, and so may explain abnormal gene expression in the presence of an apparently normal gene. One or more schizophrenia disease ‘genes’ may turn out to include abnormal transcriptional units that code for RNA regulators of protein coding gene expression or to be proximal to such units, rather than to be abnormalities in the protein coding gene itself. Regulating pseudogenes, miRNA, or other ncRNA may exist at very different chromosomal locations than the regulated gene itself. Thus, it may be that one or more areas identified through genetic linkage studies as associated with schizophrenia may in fact be the location of a regulatory ncRNA. Molecular Psychiatry Expanding the ‘central dogma’ DO Perkins et al 74 Could noncoding RNA regulate putative schizophrenia genes? The understanding of the importance of ncRNA regulation of gene expression is an emerging area of active research with many methodological challenges.5 The results of animal and plant research to date suggest that ncRNAs are likely to be a major regulatory mechanism of gene expression regulation in humans. Pertinent examples are discussed above, although numerous other ncRNA regulatory mechanisms exist.31,46,5,8 The following examples, while speculative in nature, are meant to illustrate how altered regulation of mRNA expression could potentially play a role in schizophrenia risk. Similar mechanisms could also contribute to the risk of other complex brain disorders, especially those with a neurodevelopmental etiology. ment and later in life. hsa-mir-1 maps to chromosome 20q13.33, which placed #21 in the Lewis metaanalysis, and is just distal to an area that was 11th. hsa-mir-206 maps to chromosome 6p12.2, which placed #36 in the Lewis meta-analysis, and is in between two areas with significant linkage (#2 and #5). Further study is needed to confirm whether this hypothesized relationship between miRNAs, hsa-mir1 and hsa-mir-206, is etiologically important in schizophrenia. For example, molecular genetic studies would be needed to demonstrate empirically that either hsa-mir-1 or hsa-mir-206 (or both) is simultaneously present in a cell with mRNA for BDNF, and that such miRNAs affect BDNF expression. If confirmed, further studies would also be needed to determine if miRNA regulation of BDNF is altered in schizophrenia. Micro-RNA GABA and glutamate neuronal circuits have been implicated in schizophrenia. Brain-derived neurotrophic factor (BDNF) regulates the development, survival, and synaptic maintenance of a variety of neurons in the CNS, including GABA and glutamatergic neurons47,48 BDNF has been shown to increase the survival of glutamate neurons and promote glutamate pyramidal cell dendritic growth and spine density. BDNF, through control of synaptic density, may regulate long-term potentiation of glutamate neurons. Cortical neuron dendrite growth and synapse formation is associated with an increase in cortical BDNF mRNA. In addition, BDNF may regulate type 3 dopamine receptor (DRD3) expression during brain development and promote DRD3 receptor expression in adults.49,50 Recent studies have examined BDNF expression in post-mortem brains. One study finds decreased BDNF gene expression in the dorsolateral prefrontal cortex of subjects with schizophrenia compared with healthy subjects,51 although other studies have contradictory findings.52,53 Animal models suggest that antipsychotics may impact BDNF expression,54 complicating the interpretation of these post-mortem studies. Genetic association studies between polymorphisms of the BDNF gene and schizophrenia are inconclusive, with negative55–58 and positive59,60 studies. BDNF polymorphisms have been associated with treatment responsivity61 and negative symptom severity.62 Although findings are mixed, BDNF remains a plausible candidate gene for schizophrenia. A theoretical mechanism that could explain altered mRNA expression and protein production in the presence of a normal gene is that ncRNA regulation of BDNF gene expression is disrupted. Bioinformatic investigations indicate that two newly described miRNAs, hsa-mir-1 and hsa-mir-206, may target the 30 -UTR of BDNF,22 consistent with the theory that miRNA could regulate BDNF protein synthesis by interfering with BDNF mRNA translation, potentially during brain develop- Other ncRNA The observation of an association of the genes ‘disrupted in schizophrenia 1’ (DISC1) and ‘disrupted in schizophrenia 2’ (DISC2) with schizophrenia first occurred in a family study where a balanced translocation between chromosomes 1 and 11 segregated with schizophrenia.63 This dislocation was found to disrupt the function of these two genes (hence the name). DISC1 is expressed in neurons and glia, and there is evidence that DISC1 may be involved in neuronal migration during brain development,64–66 and may modulate signal transduction and glutamate transmission.67 While several schizophrenia linkage studies found significant signal in the 1q42 region,68– 70 overall the linkage evidence for involvement of this area with schizophrenia is weak.37 DISC2 is known to be transcribed into ncRNA, but the ncRNA transcript does not code a protein product.71 The ncRNA product is partially complementary to DISC1, consistent with a regulatory role for DISC1 gene expression. Further study would be needed to determine if an actual regulatory role exists, and if DISC2 regulation of DISC1 expression is involved in schizophrenia. Molecular Psychiatry mRNA stability The type 2 dopamine receptor gene (DRD2) is a candidate gene for schizophrenia risk or disease severity due to the overwhelming evidence that schizophrenia pathology involves dysregulation of the dopamine system, especially involving DRD2 receptors.72 However, genetic studies find no or only a weak association between DRD2 gene polymorphisms and schizophrenia.73 Synonymous polymorphisms, in other words base substitutions that do not have a predicted effect on the synthesized protein, are often not examined in association studies. In particular, known DRD2 synonymous polymorphisms have not been examined in association studies because of the presumed lack of potential to impact disease.74 Recent studies suggest, however, that these synonymous polymorphisms may, in fact, not be Expanding the ‘central dogma’ DO Perkins et al functionally neutral. Synonymous DNA polymorphisms may result in different three-dimensional folding patterns of the transcribed mRNA. The altered tertiary structure of the mRNA may have consequences for mRNA degradation and translation, thus impacting rate of protein synthesis.9 In a recent study, the mRNA from one of the presumed synonymous DRD2 DNA polymorphisms SNP C957T was found to have altered tertiary structure that decreased the stability of the mRNA and consequently the rate of DRD2 protein synthesis.74 Furthermore, the SNP C957T variant DRD2 receptor was less responsive to dopamine induced upregulation. Interestingly, another synonymous polymorphism C1101A impacted mRNA translation rate, but the combination of SNPs C1101A and C957T did not. It remains to be shown whether the synonymous SNPs actually impact dopaminergic function in vivo or otherwise contributes to schizophrenia risk or disease severity. However, there are other examples where alterations in mRNA stability may be associated with brain disease, notably Alzheimer’s disease.9,4 Epigenetic evidence Factors operating independently of gene bp sequences are likely important in schizophrenia, as monozygotic twins will have an estimated concordance for schizophrenia of only 41–65%.75 Environmental factors are presumed to be important, including environmental events that occurred during brain development (eg maternal infection, starvation, obstetrical complications, maternal exposure to a disaster) and environmental exposures that occurred later in life (eg stressful life events, marijuana or stimulant drug use).76,77 Several investigators have proposed that environmental factors influence schizophrenia risk by epigenetic modification of protein coding gene expression.78–80 Converging evidence suggests that schizophrenia may be related to altered connectivity between brain regions, especially at the level of the synapse.81 There are numerous proteins involved in regulating and maintaining synaptic integrity. One such protein is Reelin (RELN), synthesized by GABA interneurons, and thought to be involved in maintaining dendritic spine density and function in neighboring pyramidal cells.78 However, the location of the RELN gene on chromosome 7q22.1 is not in a schizophrenia risk area as defined by genetic linkage studies.37 There is convincing evidence from post-mortem studies that RELN mRNA and protein expression are significantly lower in the brains of individuals with schizophrenia compared to healthy individuals. Therefore, if altered RELN expression is involved in schizophrenia, it may be due to abnormal regulation of RELN expression. A recent study suggests that the epigenetic regulation of RELN expression may be altered.78 DNA cytosine-5 methyltransferase (DNMT) is a protein critical to methylation and thus epigenetic silencing of a gene. In the same brains where RELN was underexpressed, a protein important in promot- ing epigenetic gene silencing, DNA cytosine-5 methyltransferase 1 (DNMT1), was expressed at high levels. The promoter region of the gene that codes for RELN contains the binding sites for DNMT1 (CpG islands), suggesting that epigenetic regulation is possible for the RELN gene. 75 Conclusions The recent sequencing of the human genome, advances in bioinformatics and molecular genetics, and the pervasive availability of high-speed Internet communication provide an unprecedented and readily accessible set of discovery tools. The tools have initiated and accelerated much investigation of regulation of gene expression, undoubtedly a phenomenon critical to the development and maintenance of complex life. The central dogma is now known to describe only a part of the genetic information machinery, namely what proteins might possibly be synthesized; additional genetic information controls the rates of syntheses. Recent experimental evidence suggests a role for ncRNA in other complex diseases, including cancers and fragile X syndrome. Several studies now implicate miRNAs, in particular, with cancer etiology.82 For example, in one study the levels of miRNAs hsa-mir143 and has-mir-145 were reduced in colorectal cancer cells.83 Another investigation found that the genes coding for hsa-mir-15 and hsa-mir-16, located on chromosome 13q14, were deleted or expressed at reduced levels in the tumor cells from 68% of patients with B-cell chronic lymphocytic leukemia.84 Finally, the BIC gene, containing a region that codes for hasmir-155, was overexpressed in patients with Hodgkin’s lymphoma,85 with a second study finding that both hsa-mir-155 and the BIC gene were excessively expressed in the cells from another cancer, Burkett’s lymphoma.86 The possible role of miRNA in the etiology of fragile X syndrome is suggested in an animal model that found the etiologic fragile X mental retardation protein to regulate synaptic plasticity via a mechanism that involves miRNAs.87 To date there is no evidence linking ncRNAs and schizophrenia, and so the examples provided above are highly speculative. However, elucidation of the genetic information that controls (directly or indirectly) the rate of protein synthesis might be fundamental to understanding disease expression, especially with neurodevelopmental diseases. The functions of disease risk genes might be disrupted directly through mutation of protein coding regions (that make protein production possible); indirectly through mutation of ncRNA transcription sites (that determine the rates of protein production); or through environmental impact on epigenetic regulation of gene expression. This perspective may provide the basis of new interpretations of genetic association studies, where markers may point to large genomic regions (hundreds to tens of thousands of bp’s) that are often in introns or to associations with exonic Molecular Psychiatry Expanding the ‘central dogma’ DO Perkins et al 76 regions that do not result in protein-altering mutations. Every gene implicated in the etiology of schizophrenia gives rise to multiple combinations of potential regulatory mechanisms and their associated sites of transcription. In order to focus investigative efforts, the most likely potential regulatory mechanisms must be selected. As illustrated above, investigative strategies may now include bioinformatics and may use mathematical modeling and genomic search techniques to generate testable hypotheses (eg predict new ncRNAs and their control functions, tertiary structures of mRNA, etc). While progress can be expected from existing molecular, clinical, and epidemiological methodologies, the general elucidation of the foundations of genetic regulation may be required before the genetic basis of schizophrenia and other complex disorders is fully understood. Acknowledgements This work was supported in part USPHS Grants K23MH01905-01, P50-MH064065, and UO1 MH066069 (DOP) References 1 Mattick JS. 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