CHAPTER TWO

Contribution of Long Noncoding RNAs to Autism Spectrum Disorder Risk Brent Wilkinson*,†, Daniel B. Campbell†,{,1

*Program in Biological and Biomedical Sciences, University of Southern California, Los Angeles, California, USA † Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, USA { Department of Psychiatry and the Behavioral Sciences, University of Southern California, Los Angeles, California, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5.

Small ncRNA Long ncRNA LncRNA in Fundamental Genetic Mechanisms LncRNAs in Cancer LncRNAs in the Brain 5.1 Brain development and function 5.2 Neurodegenerative diseases 5.3 Neurodevelopmental disorders 6. LncRNAs Contribute to ASD 7. Conclusions References

36 38 38 40 41 41 44 45 47 48 49

Abstract Accumulating evidence indicates that long noncoding RNAs (lncRNAs) contribute to autism spectrum disorder (ASD) risk. Although a few lncRNAs have long been recognized to have important functions, the vast majority of this class of molecules remains uncharacterized. Because lncRNAs are more abundant in human brain than proteincoding RNAs, it is likely that they contribute to brain disorders, including ASD. We review here the known functions of lncRNAs and the potential contributions of lncRNAs to ASD.

Projects aiming to characterize all of the functional elements in the mammalian genome such as ENCODE (Djebali et al., 2012) and FANTOM3 (Carninci et al., 2005) have made significant strides in our understanding of the complexity of transcriptional regulation. It is now clear that while less

International Review of Neurobiology, Volume 113 ISSN 0074-7742 http://dx.doi.org/10.1016/B978-0-12-418700-9.00002-2

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2013 Elsevier Inc. All rights reserved.

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than 2% of the mammalian genome codes for proteins, a majority of what used to be thought of as “junk DNA” actually undergoes transcription and produces noncoding RNAs (ncRNAs) (Bertone et al., 2004; Birney et al., 2007; Carninci et al., 2005, 2006; Cheng et al., 2005; Core, Waterfall, & Lis, 2008; Djebali et al., 2012; Kapranov et al., 2007; Lander et al., 2001; Seila et al., 2008). Emergence of the functional characteristics of ncRNA has shown that these regulatory RNAs play several critical roles in modulating gene expression and influence the developmental complexity of higher species. Considering the proportion of ncRNA relative to total genome size, there is a significant correlation between increased ncRNA in a species with developmental complexity (Taft, Pheasant, & Mattick, 2007). Because of this role, it can be inferred that ncRNA will take part in many critical functions in the most complex organ of the body, the brain. NcRNA can be broadly categorized into two classes based on their size: short ncRNAs (200 nucleotides in length) (Kapranov et al., 2007), both of which contain a number of diverse subclasses with their own distinct properties. Currently, relatively few ncRNAs have been functionally characterized compared to the large amount shown to undergo transcription. But for those that have, it is clear that many are integral to the processes of development and maintenance of an organism. Being essential components of regulatory networks within our cells also means that when they become aberrant, they can influence disease. Indeed, ncRNAs have been implicated in a number of conditions including cancer, HIV, heart disease, and neurological disorders. This highlights the critical importance of characterizing ncRNAs which may provide a pathway to discover routes of pathogenesis that are currently unknown and improve on those that are. Here, we outline the current progress of characterizing ncRNAs and their relationship to several neurological disorders. Specifically, we show that a number of ncRNAs have been identified in autism spectrum disorder (ASD) and associated neurodevelopmental disorders, may play critical roles in these, and may serve as potential therapeutic targets.

1. SMALL ncRNA The classification, small ncRNA, encompasses a wide variety of subclasses including microRNAs (miRNAs), short interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), and PIWI-interacting RNAs (piRNAs). These can be derived from a variety of sources within the

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genome (review in Mattick & Makunin, 2005) and it has been suggested that one potential source is also from the processing of long noncoding RNAs (lncRNAs) (Jalali, Jayaraj, & Scaria, 2012; Keniry et al., 2012). In general, one of the most prominent forms of genetic regulation carried out by small ncRNAs is by binding to their specific target transcripts via complementary nucleotide sequences and subsequently influencing the translation of the target through a diverse set of downstream events. For an in-depth explanation of small ncRNA, including subclasses, biogenesis, and forms of genetic regulation, we refer the reader to these reviews: Carthew and Sontheimer (2009), Kim, Han, and Siomi (2009), Luteijn and Ketting (2013), Matera, Terns, and Terns (2007), Mattick and Makunin (2005), and Winter, Jung, Keller, Gregory, and Diederichs (2009). One of the most thoroughly studied classes of small ncRNAs, miRNAs, was first discovered in Caenorhabditis elegans in 1993 (Lee, Feinbaum, & Ambros, 1993) and since then has been postulated to be relatively conserved among vertebrate species (Altuvia et al., 2005; Berezikov et al., 2005; Iba´n˜ez-Ventoso, Vora, & Driscoll, 2008). MiRNAs can participate in posttranscriptional gene regulation by binding to either the 30 -UTR (Lai, 2002) or the 50 -UTR (Lytle, Yario, & Steitz, 2007) of a target transcript which subsequently inhibits its translation. A single miRNA has the potential to target multiple transcripts and similarly, a given transcript may be regulated by more than one miRNA (Krek et al., 2005). Both the importance and prevalence of miRNAs in the human genome is underscored by the fact that greater than 60% of human protein-coding genes are predicted targets of miRNAs (Friedman, Farh, Burge, & Bartel, 2009). The regulation performed by miRNAs is a critical process in the development of mammals and like many other ncRNAs, their expression can be specific in both particular tissue types and developmental stages as reviewed in AlvarezGarcia and Miska (2005), Sayed and Abdellatif (2011), and Wienholds and Plasterk (2005). Like miRNA, siRNA also regulates gene expression by binding to complementary sequences on target transcripts, but with more stringent requirements as they have to have close to perfect sequence complementation (Elbashir, Martinez, Patkaniowska, Lendeckel, & Tuschl, 2001). In general, this difference results in two separate mechanisms where targets with close to perfect complementation undergo direct cleavage (as with siRNA) and those with relatively loose complementation are destabilized or transcriptionally repressed (as with miRNA) (Guo, Ingolia, Weissman, & Bartel, 2010; Hutva´gner & Zamore, 2002; Martinez, Patkaniowska, Urlaub,

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Lu¨hrmann, & Tuschl, 2002). Endogenous siRNAs (endo-siRNAs) in Drosophila have been proposed to protect against dsDNA viruses and transposable elements in somatic tissues (Chung, Okamura, Martin, & Lai, 2008; Li, Li, & Ding, 2002; Wang et al., 2006). Endo-siRNAs have also been identified in human cells (Chan et al., 2013; Xia, Joyce, Bowcock, & Zhang, 2013; Yang & Kazazian, 2006) and in mice (Babiarz, Ruby, Wang, Bartel, & Blelloch, 2008; Song et al., 2011; Tam et al., 2008; Watanabe et al., 2008), showing another layer of genetic regulation in mammals. siRNA has been explored in numerous settings as a therapeutic agent used for gene therapy and has potential for correcting the dysregulation of ncRNA in several different diseases (Burnett & Rossi, 2012).

2. LONG ncRNA LncRNA is a broad category which encompasses transcripts of diverse structural features and mechanisms of action. They can be derived from sense or antisense strands overlapping with protein-coding genes, within intergenic regions (lincRNAs), or within pseudogenes. Once transcribed they may undergo splicing and be processed to include a 50 methyl-guanosine cap and 30 -poly (A) tail. Within the cell, lncRNAs can be localized to the nucleus or the cytoplasm (Ponting, Oliver, & Reik, 2009). They have been found to perform a wide variety of functions including participating in the recruitment of chromatin-modifying complexes, acting as competing endogenous RNAs (ceRNAs), providing a scaffold for the assembly of protein complexes, modulating alternative splicing, and employing enhancer-like functions (Nagano & Fraser, 2011; Rinn & Chang, 2012; Wang & Chang, 2011). This diverse set of functions further emphasizes the notion that lncRNAs are key components of numerous cellular processes (Table 2.1).

3. LncRNA IN FUNDAMENTAL GENETIC MECHANISMS X-chromosome inactivation (XCI) is essential to the identity of female mammals and is a highly intricate process involving multiple lncRNAs that work together in order to ultimately downregulate mass quantities of genes for dosage compensation. X-inactive specific transcript (Xist) is a conserved lncRNA specifically expressed in the inactive X-chromosome and required for XCI (Brown et al., 1992; Brown, 1991). This was confirmed in XX murine embryonic stem cells (mESCs)

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Table 2.1 LncRNAs implicated in neurodevelopmental and neurodegenerative disorders lncRNA Function Reference

ASFMR1

Unknown, co-expressed with FMR1 Ladd et al. (2007)

ATXN8OS Unknown

Moseley et al. (2006)

BACE1-AS Regulation of BACE1 and roles in amyloid plaque formation

Faghihi et al. (2008)

BC200

Initiation of protein translation in dendritic processes

Muddashetty et al. (2002), Mus, Hof, and Tiedge (2007)

BDNFOS

Regulation of BDNF

Lipovich et al. (2012)

DISC2

Unknown, antisense to DISC1

Millar et al. (2000)

EVF2

Involvement in GABAergic interneuron function

Bond et al. (2009), Feng et al. (2006)

FMR4

Antiapoptic functions, co-expressed with FMR1

Khalil, Faghihi, Modarresi, Brothers, and Wahlestedt (2008)

Gomafu

Alternative splicing of DISC1 and ERBB4

Barry et al. (2013)

HAR1F

Unknown, expressed in Cajal-Retzius Pollard et al. (2006) neurons of developing human neocortex

MALAT1

Regulation of alternative splicing and Bernard et al. (2010) synaptogenesis

MSNP1AS Regulation of Moesin

Kerin et al. (2012)

SOX2OT

Neurogenesis

Amaral et al. (2009)

UBE3AATS

Genomic imprinting of UBE3A

Meng, Person, and Beaudet (2012)

by introducing a targeted deletion of Xist into a single allele. Following differentiation, the targeted X chromosome would always fail to inactivate and only the X chromosome expressing Xist would undergo XCI (Penny, Kay, Sheardown, Rastan, & Brockdorff, 1996). Xist acts by coating the chromosome to be inactivated and then recruiting polycomb-group proteins for inactivation via epigenetic mechanisms (Plath et al., 2003). By manipulating the expression of Xist, selective silencing of one copy of chromosome 21 was achieved in induced pluripotent stem cells (iPSCs) derived from patients

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with Down’s syndrome. In the iPSC model, this corrected for the trisomy of chromosome 21 involved in the pathogenesis of Down’s syndrome and improved deficiencies in proliferation and neural rosette formation (Jiang et al., 2013). In addition, it was recently discovered that there is also a human specific lncRNA, XACT, that coats the active X chromosome in pluripotent cells (Vallot et al., 2013). Xist displays a common trait among lncRNAs as they can associate with chromatin and recruit proteins with epigenetic functions in order to regulate the transcription of multiple genes. LncRNAs have also been shown to be involved in genomic imprinting, the process by which genes are expressed in a parent-specific manner. The paternally expressed lncRNA, Antisense Igf 2r (Air), silences three proteincoding genes (Igf2r, Slc22a2, and Slc22a3) located within the Igf2r cluster on the same allele (Sleutels, Zwart, & Barlow, 2002). Like Xist, Air is conserved between mice and humans (Yotova et al., 2008) and is involved in epigenetic regulation as it inactivates the Slc22a3 gene by recruiting G9a (a histone methyltransferase) (Nagano et al., 2008). Factors influencing genomic imprinting are of extreme importance as dysregulation of this process is hallmark of a number of conditions, including the neurodevelopmental disorders, Angelman Syndrome (AS), and Prader-Willi Syndrome (PWS) (Horsthemke & Wagstaff, 2008).

4. LncRNAs IN CANCER Downregulation of the tumor suppressor, PTEN, has been associated with numerous types of cancers (Cairns et al., 1997; Li et al., 1997; Vlietstra, van Alewijk, Hermans, van Steenbrugge, & Trapman, 1998). The lncRNA, PTENpg1 (also known as PTENP1), is selectively lost in cancer, has similar expression levels to that of PTEN, and functions as an miRNA decoy by intercepting miRNA species targeting PTEN (Poliseno et al., 2010). Two PTENpg1 antisense (as) isoforms, a and b, regulate the expression levels of PTEN by diverse mechanisms, illustrating the complex networks of regulation lncRNAs can take part in. PTENpg1 asRNA a is able to decrease PTEN expression levels through epigenetic mechanisms involving DNMT3A and EZH2, while PTENpg1 asRNA b interacts with PTENpg1 in order to positively influence its stability and decoy function ( Johnsson et al., 2013). Interestingly, abnormalities in PTEN have also been implicated in ASD (Butler et al., 2005; O’Roak et al., 2012). The HOX genes are essential for specifying patterning during the development of bilateral animals (Pearson, Lemons, & McGinnis, 2005).

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HOTAIR is an lncRNA transcribed from the HOXC locus that acts in trans to regulate transcription at the HOXD locus. Here, HOTAIR associates with Polycomb Repressive Complex 2 (PRC2) in order to influence Histone H3 Lysine-27 trimethylation which results in transcriptional silencing (Rinn et al., 2007). Increased expression of HOTAIR has been associated with a variety of cancer types including breast (Gupta et al., 2010), colon (Kogo et al., 2011), and liver (Ishibashi et al., 2013) cancers. This may be due to massive epigenetic changes in which increased HOTAIR reverts the epigenetic profile of cancerous cells to something resembling that of embryonic fibroblasts through retargeting of PRC2 (Gupta et al., 2010). Maternally expressed gene 3 (MEG3) is a conserved, imprinted gene encoding an lncRNA. In meningiomas, there is a strong association between loss of MEG3 expression and increase in tumor grade (Zhang et al., 2010). This can be attributed to the activation of the p53 pathway (Zhao, Dahle, Zhou, Zhang, & Klibanski, 2005) and increased methylation of MEG3 regulatory region. The effect of decreased expression of MEG3 has also been implicated in pituitary adenomas (Zhang et al., 2003), gliomas (Wang, Ren, & Sun, 2012), cervical cancer (Qin et al., 2013), and bladder cancer (Ying et al., 2013).

5. LncRNAs IN THE BRAIN LncRNAs have been shown to be highly expressed within the central nervous system and in particular, the brain, where they can exhibit spatiotemporal expression patterns (Lipovich et al., 2013; Ponjavic, Oliver, Lunter, & Ponting, 2009). This highly diverse class of ncRNAs has been shown to be involved in several key roles of brain development and function. Therefore, dysregulation of lncRNAs can be contributing factors to neurological disorders, which we will show examples with respect to neurodevelopmental and neurodegenerative disorders. For many complex neurological disorders such as autism, the pathogenesis is quite unclear. The functional characterization of lncRNAs could unveil another layer of transcriptional regulation involved in their pathogenesis and potentially provide routes of therapeutic intervention.

5.1. Brain development and function LncRNAs are essential to the development, maintenance, and function of the brain. They have been shown to take part in fundamental processes such as synaptogenesis, neurogenesis, and GABAergic interneuron function.

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Abnormalities in these processes have been implicated in several neurodevelopmental disorders including ASD and schizophrenia (SZ). Dysregulation of lncRNAs involved in these processes may ultimately impact the molecular mechanisms that underlie the observed phenotypes in these disorders. Neurogenesis, the differentiation of neurons from neural stem cells or neural progenitor cells, is a critical process that occurs throughout the life of an individual (Ming & Song, 2005). Studies analyzing the differential expression of lncRNAs upon differentiating human ESCs or iPSCs to neurons have identified several lncRNAs as integral components of neurogenesis (Lin et al., 2011; Ng, Johnson, & Stanton, 2012). The proteincoding gene, SOX2, has been shown to play key roles in both embryonic and adult neurogenesis (Ellis et al., 2004; Favaro et al., 2009; Ferri et al., 2004) and is located within an intron of the lncRNA Sox2 overlapping transcript (SOX2OT) (Fantes et al., 2003). Sox2ot and an alternatively spliced isoform, Sox2dot, are both expressed in the mouse brain and enriched in areas associated with neurogenesis (Amaral et al., 2009; Mercer, Dinger, Sunkin, Mehler, & Mattick, 2008). Defects in neurogenesis during development and adulthood have been linked to a number of neurodevelopmental and neurodegenerative diseases (Amiri et al., 2012; Guidi et al., 2008; Hsieh & Eisch, 2010; Reif et al., 2006; Wegiel et al., 2010). In the human brain, synaptogenesis is characterized by increased proliferation of neuronal cells and an overproduction of synaptic connections from gestation to about 3 years of age, followed by subsequent “trimming” of these into adulthood (Bourgeron, 2009; Huttenlocher & Dabholkar, 1997; Petanjek et al., 2011). This timeline has many important implications in neurodevelopmental disorders, as ASD typically presents itself prior to the age of 3 (Bourgeron, 2009; Investigators & Prevention, 2012). Metastasisassociated lung-adenocarcinoma transcript 1 (MALAT1) was originally identified as being overexpressed in non-small cell lung cancer ( Ji et al., 2003) and is also highly expressed in neurons where it plays key roles in synaptogenesis. This lncRNA was shown to regulate synaptic density and the expression levels of neuroligin1 (NLGN1) and synaptic cell-adhesion molecule (SynCAM1), which are involved in controlling synapse formation (Bernard et al., 2010). As with neurogenesis, aberrant regulation of synaptogenesis is a common theme among many neurological disorders including ASD and SZ (Bourgeron, 2009; Grant, 2012; Zoghbi, 2003). In addition, MALAT1 takes part in the recruitment of SR-type pre-mRNA splicing factors to nuclear speckle domains where they participate in

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regulating alternative splicing (Tripathi et al., 2010). This is particularly relevant to the human brain, which contains one of the highest proportions of alternatively spliced transcripts (de la Grange, Gratadou, Delord, Dutertre, & Auboeuf, 2010; Grosso et al., 2008). Dysregulation of alternative splicing can impact the expression levels of large quantities of transcripts and has been implicated in SZ and ASD (Morikawa & Manabe, 2010; Voineagu et al., 2011). GABA is one of the most abundant neurotransmitters in the brain and has key roles in development (Wonders & Anderson, 2006). Because of this, irregular GABAergic interneuron function has been linked to ASD (Fatemi, Folsom, Kneeland, & Liesch, 2011; Hogart, Nagarajan, Patzel, Yasui, & Lasalle, 2007; Horder et al., 2013) and SZ (Lewis, Hashimoto, & Volk, 2005). Dlx homeobox genes are critical for the differentiation and migration of GABAergic interneurons in the developing brain (Anderson, Eisenstat, Shi, & Rubenstein, 1997; Kuwajima, Nishimura, & Yoshikawa, 2006). Evf2 is an lncRNA transcribed from the Dlx-5/6 ultraconserved region that recruits DLX and MECP2 transcription factors to this same region in order to influence the expression of Dlx5, Dlx6, and Gad1. This occurs through a combination of both cis and trans mechanisms (Bond et al., 2009) and illustrates how lncRNAs are able to regulate specific targets through diverse mechanisms. Evf2 knockout mice show reduced numbers of GABAergic interneurons in the hippocampus and dentate gyrus during infancy. Although the quantity of interneurons returns to normal in adults, defects in synaptic connectivity remain (Bond et al., 2009). The early dysregulation in Evf2 having a long-lasting influence highlights the importance of lncRNA in neurodevelopmental disorders. BDNF, the most abundant neurotrophin in the brain, takes part in several fundamental functions including the regulation of neuron morphology, neuronal cell survival, and neuronal plasticity and memory (Egan et al., 2003; Horch & Katz, 2002; Tanaka et al., 2008). Due to its diverse roles in fundamental processes, BDNF has been linked to a wide range of neurodevelopmental and neurodegenerative disorders including SZ (Krebs et al., 2000; Neves-Pereira et al., 2005; Nieto, Kukuljan, & Silva, 2013), Alzheimer’s disease (AD) (Hock, Heese, Hulette, Rosenberg, & Otten, 2000), Parkinson’s disease (Howells et al., 2000), and ASD (Gadow, Roohi, DeVincent, Kirsch, & Hatchwell, 2009; Katoh-Semba et al., 2007; Ricci et al., 2013). BDNFOS or anti-BDNF is a conserved natural antisense transcript that forms dsRNA duplexes in the brain in order to downregulate BDNF transcript levels (Liu et al., 2006; Pruunsild,

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Kazantseva, Aid, Palm, & Timmusk, 2007). In neocortical regions of brain tissue removed from patients to treat intractable seizures, an increase in BDNF expression along with a reciprocal decrease in BDNFOS expression has been observed (Lipovich et al., 2012). This same trend was seen in human neuronal cells after repeated depolarization that mimicked the effects of epileptic seizures. In addition, the lncRNAs RPPH1, NEAT1, and MALAT1 were also upregulated in both experimental settings, showing that lncRNAs can have activity-dependent expression (Lipovich et al., 2012).

5.2. Neurodegenerative diseases In addition to contributing to the fundamental processes of development, lncRNAs can also be causative factors in diseases characterized by rapid decline of the brain. BACE1 is involved in cleaving APP in order to generate the toxic Ab peptides that contribute to the pathogenesis of AD (Cai et al., 2001) and decreased expression of BACE1 leads to reduced levels of Ab (Atwal et al., 2011; Singer et al., 2005). BACE1-antisense transcript (BACE1-AS) is upregulated in both a transgenic mouse model and human patients with AD. With increased BACE1-AS, there is a concurrent upregulation of BACE1 which is proposed to be due to stabilization of BACE1 by BACE1-AS (Faghihi et al., 2008). This is in contrast to the mechanisms proposed for other protein-coding genes and their corresponding antisense transcripts such as BDNF/BDNFOS and UBE3A/UBE3-ATS where the antisense transcript imposes downregulation of the protein-coding gene (Lipovich et al., 2012; Meng et al., 2012). Upon exposure to Ab 1–42, expression of both BACE1 and BACE1-AS increases which subsequently acts to further increase the levels of Ab 1–42. This can lead to the formation of amyloid plaques and progression of AD (Faghihi et al., 2008). BC200 RNA is a brain-specific ncRNA that is homologous to the rodent BC1 RNA (Martignetti & Brosius, 1993; Tiedge, Chen, & Brosius, 1993). In the dendritic processes of neurons, BC200 RNA associates with poly(A)-binding protein (PABP1) and plays a role in regulating the initiation of protein translation (Muddashetty et al., 2002). Expression of BC200 RNA is upregulated in selectively vulnerable areas of the brain in AD. This upregulation increases with severity of AD and skews the distribution of BC200 RNA, causing it to be clustered in the cell soma (Mus et al., 2007).

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5.3. Neurodevelopmental disorders Silencing of the Fragile X Mental Retardation Gene (FMR1) resulting from extensive 50 -UTR CGG trinucleotide repeats is a causative factor in the most common form of inherited mental retardation, fragile X syndrome (FXS). Normal FMR1 genes contain 5–54 repeats, 55–200 repeats is categorized as the premutation allele, and above 200 repeats is termed the full mutation allele and has a direct link with FXS. The premutation allele confers increased expression of FMR1 mRNA and is associated with fragile X-associated tremor and ataxia syndrome (FXTAS) while the full mutation allele silences the transcription of FMR1 (Garber, Visootsak, & Warren, 2008). Two lncRNAs, ASFMR1 and FMR4, are transcribed from the FMR1 locus and have been shown to have similar patterns of expression with respect to FMR1 in both the premutation and full mutation alleles. Although the function of ASFMR1 is currently unknown, FMR4 has been shown to have antiapoptotic properties in vitro (Khalil et al., 2008; Ladd et al., 2007). In addition, carriers of the premutation allele have been associated with mitochondrial dysfunction (Ross-Inta et al., 2010), which may predispose individuals to neurodegenerative disorders such as Parkinson’s disease (Loesch et al., 2011). Based on the similar expression patterns of both ASFMR1 and FMR4, there is a significant possibility of their contribution to the pathogenesis of the FXS and FXTAS. Like ASD, SZ is a complex disorder thought to be the result of a variety of genetic and environmental influences (Sullivan, Kendler, & Neale, 2003). The DISC locus was originally identified in a large Scottish family as a candidate gene for SZ and contains DISC1 along with a human-specific lncRNA antisense to DISC1 and DISC2 (Millar et al., 2000; Taylor, Devon, Millar, & Porteous, 2003). Recently, it was shown that the lncRNA, Gomafu, is involved in the alternative splicing of DISC1 and another SZ-associated gene, ERBB4. Gomafu is downregulated in an activity-dependent manner in response to depolarization in both mouse and human neuronal cell lines. Knockdown of Gomafu results in increased expression of alternatively spliced isoforms of DISC1 and ERBB4 but not the unspliced genes; this is the same expression pattern previously observed in postmortem brain tissue of schizophrenic patients (Barry et al., 2013; Law, Kleinman, Weinberger, & Weickert, 2007; Nakata et al., 2009). Importantly, decreased expression of Gomafu was also observed in postmortem brain tissue of individuals with SZ, implicating this lncRNA in the pathogenesis of SZ and as a potential therapeutic target (Barry et al., 2013).

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UBE3A-ATS is an lncRNA antisense to UBE3A within the 15q11–13 chromosomal region (Rougeulle, Cardoso, Fonte´s, Colleaux, & Lalande, 1998), which is termed the PWS/AS region due to chromosomal abnormalities arising here being causative factors to these disorders (Nicholls & Knepper, 2001). The failure to inherit/express a maternal copy of UBE3A is known to cause AS in a majority of the cases (Kishino, Lalande, & Wagstaff, 1997). UBE3A is imprinted in a neuron-specific manner, where it is only expressed from the maternal allele due to silencing of UBE3A on the paternal allele by the lncRNA, UBE3A-ATS (Meng et al., 2012; Yamasaki et al., 2003). This suggests UBE3-ATS may also be a useful therapeutic target as dysregulation of UBE3A-ATS can cause abnormal expression of paternal UBE3A and contribute to the pathogenesis of AS. Due to the tissue- and cell type-specific expression of many lncRNAs, they have the potential to be utilized as biomarkers in order to monitor developmental changes within the brain. Rhabdomyosarcoma 2-associated transcript (RMST ) was originally identified as differentially expressed in various tumor types (Chan, Thorner, Squire, & Zielenska, 2002). It has subsequently been shown that the expression of this conserved lncRNA is mainly restricted to the CNS (Chodroff et al., 2010) and is a marker for midbrain dopaminergic neurons in mice (Uhde, Vives, Jaeger, & Li, 2010). Although a function in human neurons is yet to be elucidated, a recent genome-wide association study identified RMST as a risk gene for severe obesity in which the other loci having significant association were involved in neuronal regulation of energy homeostasis (Wheeler et al., 2013). While some lncRNAs are conserved among species, a majority of these are not in comparison to the prevalence of conserved protein-coding genes (Mercer, Dinger, & Mattick, 2009; Pang, Frith, & Mattick, 2006). Those that are human specific may be linked to human complexity and elucidate pathways involved in complex disorders. By comparing the human genome against that of the chimpanzee and looking for regions that display accelerated evolutionary change, Pollard et al. identified “human accelerated region” (HAR1). HAR1 is part of a pair of overlapping transcripts termed HAR1F and HAR1R. HAR1F displays both time- and cell type-specific expression in Cajal–Retzius neurons of the developing human neocortex that is not detectable past 24 gestational weeks (Pollard et al., 2006). This developmental specificity, or lncRNAs with expression patterns like this, could be important in neurodevelopmental disorders that have defects present from early on such as ASD or SZ. In fact, within the Cajal–Retzius neurons, HAR1F is coexpressed with reelin (Pollard et al., 2006), which has

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been implicated in SZ (Eastwood & Harrison, 2006; Impagnatiello et al., 1998) and ASD (Ashley-Koch et al., 2007; Persico et al., 2001). The involvement of lncRNA in contributing to the pathogenesis of neurodevelopmental disorders is limited to a handful of examples. But, these examples do show that lncRNAs play critical roles in human development and emphasize the need for further characterization of others. The fact that several of these disorders can have comorbid diagnoses with one another points to the possibility of some common molecular pathways that could involve lncRNA. For example, some patients diagnosed with AS also have mutations in the MECP2 gene responsible for a majority of Rett Syndrome cases (Samaco, Hogart, & LaSalle, 2005; Watson et al., 2001).

6. LncRNAs CONTRIBUTE TO ASD Direct evidence for a contribution of lncRNAs to ASD continues to accumulate. Differential expression of lncRNAs has been observed in both postmortem brain tissue and lymphoblastoid cell lines. Recently, we reported that a genome-wide significant association signal implicated an lncRNA, not the neighboring protein-coding genes. To date there has only been one study characterizing lncRNA expression profiles in postmortem tissues of individuals with ASD. Ziat and colleges detected over 222 differentially expressed lncRNAs between individuals with ASD compared to controls. Within these 222 lncRNAs, 82 were unique to the prefrontal cortex (PFC), while 143 were unique to the cerebellum (Ziats & Rennert, 2013). This observation again underlies the fact that lncRNA expression can be highly tissue specific. They also reported increased transcriptional homogeneity between the PFC and cerebellum of ASD brain tissue when compared to controls in both mRNA and annotated lncRNA (1375 differentially expressed lncRNAs in control samples versus 236 in the ASD samples) (Ziats & Rennert, 2013). Although the conclusions drawn from these results are exciting, the sample size of brain tissue in this study is relatively small (n ¼ 2 ASD patients and n ¼ 2 age, sex-matched controls). More studies of this nature will have to be repeated with a larger sample size in order to assess variability and determine the significance of these conclusions. A scan for differential expression of transcripts in LCLs derived from three subgroups of individuals diagnosed with ASD identified 20 common lncRNAs that were dysregulated in all of the subgroups compared to controls. A majority of the lncRNAs identified were also shown to be

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androgen-responsive, suggesting a gender-specific component (Hu, 2013; Hu et al., 2009). A genome-wide association study (GWAS) of ASD indicated genomewide significant association (P ¼ 1010) of rs4307059 on chromosome 5p14.1 (Wang et al., 2009). The same rs4307059 allele was also associated with social communication phenotypes in a general population sample (St Pourcain et al., 2010). However, rs4307059 genotype was not correlated with expression of either of the flanking protein-coding genes, CDH9 and CDH10 (Wang et al., 2009). We identified a 3.9 kb noncoding RNA that is transcribed directly at the site of the chromosome 5p14.1 ASD GWAS peak (Kerin et al., 2012). The noncoding RNA is encoded by the opposite (antisense) strand of moesin pseudogene 1 (MSNP1) and is thus designated MSNP1AS (moesin pseudogene 1, antisense). MSNP1AS is 94% identical and antisense to the X chromosome transcript MSN, which encodes a protein (moesin) that regulates neuronal architecture and immune response. Expression of MSNP1AS in postmortem temporal cortex is increased 12.7-fold in individuals with ASD and increased 22-fold in individuals with the rs4307059 risk allele (Kerin et al., 2012). The MSNP1AS noncoding RNA binds MSN and its overexpression in cultured neurons causes significant decreases in MSN transcript, moesin protein, neurite number, and neurite length. Thus, our discovery reveals a functional lncRNA which, based on the GWAS findings, contributes to ASD risk (Fig. 2.1).

7. CONCLUSIONS The Central Dogma of Molecular Biology posits that DNA is transcribed into RNA, which is translated into protein. Genetic information is stored in protein-coding genes, while RNA is merely an intermediary between genes and functional proteins. Prior to the completion of the Human Genome Project, the prevailing hypothesis was that the human genome would produce approximately 100,000 protein-coding genes. This seemed like a reasonable estimate based on the size of the human genome and the complexity of human anatomy. However, the human genome contains only approximately 21,000 protein-coding genes, slightly more than a mouse and slightly less than a grape. Over the last decade, RNA has become increasingly recognized as a functional entity. The whole genome and transcriptome sequencing suggests that the complexity of an organism may be regulated by noncoding portions of the genome rather than by proteins.

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A

B

C

rs7704909

rs4307059

MSNP1: Chr 5p14.1 pseudogene with 94% sequence identity to Chr X gene MSN

+ Strand – Strand MSNP1AS: Chr 5p14.1 transcribed 3.9 kb non-coding anti-sense RNA

Figure 2.1 MSNP1AS maps within the chromosome 5p14.1 GWAS-significant ASDassociation peak and is the only significantly expressed transcript within 500 kb of the GWAS peak. (A) The GWAS results from Wang et al. (2009), indicating ASDassociated markers on chromosome 5p14.1. (B) Genome-wide RNA-Seq data from a variety of tissue sources indicate that a single major transcript of 4 kb is expressed within 500 kb of the GWAS peak. (C) The þ strand of this 4 kb chromosome 5p14.1 region is the pseudogene moesin-like 1 (MSNP1), which has 94% sequence identity to the X chromosome gene-encoding moesin (MSN) but does not appear to be transcribed. Instead, our data indicate that a 3.9 kb RNA is transcribed from the  strand, producing a non-protein-coding RNA that is antisense to the X chromosome geneencoding moesin (MSN). Because the chromosome 5p14.1 transcript represents the antisense of the pseudogene, we designate it MSNP1AS (moesin pseudogene 1, antisense). Data from the UCSC Genome Browser.

Among long RNAs produced in the human brain, the majority do not code for proteins but are instead lncRNAs. Increasing evidence suggests that these lncRNAs may contribute to brain disorders.

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