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RNA is widely transcribed from a variety of genomic regions, and extensive studies on the functional roles and regulations of noncoding RNAs including antisense RNAs and small RNAs are in progress. In addition, the human genome project revealed that we humans carry as few as ∼22,000 genes. Humans exploit tissue-specific and developmental stage-specific alternative splicing to generate a large variety of molecules in specific cells at specific developmental stages. Neurological disorders are also subject to aberrations of the splicing mechanisms. This review focuses mostly on splicing abnormalities due to pathological alterations of splicing cis- elements and trans-factors. Pathomechanisms associated with disrupted splicing cis-elements can be applied to any human diseases, and we did not restrict the descriptions to neurological diseases. On the other hand, we limited the descrip- tions of dysregulated splicing trans-factors to neurological disorders. Neurological diseases covered in this review include congenital myasthenic syndromes, spinal muscular atrophy, myotonic dystrophy, Alzheimer’s disease, frontotemporal demen- tia with Parkinsonism linked to chromosome 17, facioscapulohumeral muscular dystrophy, fragile X-associated tremor/ataxia s yndrome, Prader–Willi syndrome, Rett syndrome, spinocerebellar atrophy type 8, and paraneoplastic neurological disorders. Keywords The RNA world · Pre-mRNA splicing · Splicing cis-elements · Splicing trans-factors · Branch point sequence (BPS) · Exonic splicing enhancer (ESE) · Exonic splicing silencer (ESS) · Intronic splicing enhancer (ISE) · Intronic splicing silencer (ISS) · Nonsense-mediated mRNA decay (NMD) · Nonsense-associated skipping of a remote exon (NASRE) · Congenital myasthenic syndromes · Spinal muscular atrophy (SMA) · Myotonic dystrophy (DM1, DM2) · Alzheimer’s disease· Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) · K. Ohno (B) Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan e-mail: ohnok@med.nagoya-u.ac.jp 399 J.P. Blass (ed.), Neurochemical Mechanisms in Disease, Advances in Neurobiology 1, DOI 10.1007/978-1-4419-7104-3_14, C Springer Science+Business Media, LLC 2011 400 K. Ohno and A. Masuda Facioscapulohumeral muscular dystrophy (FSHD) · Fragile X-associated tremor/ ataxia syndrome (FXTAS) · Prader–Willi syndrome, Rett syndrome · Spinocerebellar atrophy type 8 (SCA8) · Paraneoplastic neurological disorders (PND) Contents 1 Introduction 400 2 Physiology of Splicing Mechanisms 401 3 Disorders Associated with Disruption of Splicing Cis-Elements 402 3.1 Aberrations of the 5 Splice Sites 402 3.2 Human Branch Point Consensus Sequence 403 3.3 Ectopic AG Dinucleotide Abrogates the AG-Scanning Mechanism 404 3.4 Mutations That Disrupt ESE and ESS 404 3.5 Mutations That Disrupt ISE and ISS 405 3.6 Spinal Muscular Atrophy (SMA) 405 4 Skipping of Multiple Exons Caused by a Single Splicing Mutation 406 4.1 Skipping of Multiple Contiguous Exons 406 4.2 Nonsense-Associated Skipping of a Remote Exon (NASRE) 406 5 Disorders Associated with Dysregulation of Splicing Trans-Factors 407 5.1 Myotonic Dystrophy 407 5.2 Alzheimer’s Disease (AD) and Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17 (FTDP-17) 409 5.3 Facioscapulohumeral Muscular Dystrophy (FSHD) 409 5.4 Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) 410 5.5 Prader–Willi Syndrome (PWS) 410 5.6 Rett Syndrome 410 5.7 Spinocerebellar Ataxia Type 8 (SCA8) 411 5.8 Paraneoplastic Neurological Disorders (PND) 411 References 412 1 Introduction The central dogma first enunciated by Francis Crick depicts RNA as an intermedi- ate that links DNA and protein (Crick, 1970). The beginning of life, however, was the RNA world where there were no DNA or proteins (Gilbert, 1986). In the RNA world, RNA was the only carrier of genetic information that DNA currently serves as, and the only f unctional molecule that proteins currently serve as. Although the RNA transmits no genetic information to progeny and constitutes a limited num- ber of functional molecules in our human body, the RNA world is still in effect in our body. Humans transcribe more than half of our entire genome including noncoding regions. The transcripts work as antisense RNAs, microRNAs, and snoRNAs. Researchers are now working to disclose the functional significance of these noncoding RNAs. RNA Pathologies in Neurological Disorders 401 The human genome project and the subsequent annotation efforts revealed that we humans carry as few as 22,000 genes. Tissue-specific and developmental stage- specific splicing enables to us to generate more than 100,000 molecules from a limited number of genes (Black, 2003; Licatalosi and Darnell, 2006). Small RNA molecules and RNA splicing mechanisms potentially become targets of neurolog- ical diseases (Ranum and Cooper, 2006). This review focuses mostly on splicing aberrations associated with neurological disorders. 2 Physiology of Splicing Mechanisms In higher eukaryotes, pre-mRNA splicing is mediated by degenerative splicing cis- elements comprised of the branch point sequence (BPS), the polypyrimidine tract (PPT), the 5 and 3 splice sites, and exonic/intronic splicing enhancers/silencers (Fig. 1). Stepwise assembly of the spliceosome starts from recruitment of U1 snRNP to the 5 splice site, SF1 to the BPS, U2AF65 to the PPT, and U2AF35 to the 3 end of an intron to form a spliceosome complex E (Sperling et al., 2008). SF1, a 75 kDa protein, is a mammalian homologue of yeast BBP (branch point-binding protein). U2AF65 and U2AF35 bring U2 snRNP to the BPS in place of SF1 (Wu et al., 1999; Zorio and Blumenthal, 1999). The BPS establishes base pairing interactions with a stretch of “GUAGUA” of U2 snRNA (Arning et al., 1996; Abovich and Rosbash, 1997), which then bulges out the branch site nucleotide, usually an adenosine to form a spliceosome complex A (Query et al., 1994). Thereafter, pre-mRNAs are spliced in two sequential transesterification reactions mediated by the spliceosome. In the first step, the 2 -OH moiety of the branch site nucleotide carries out a nucle- ophilic attack against a phosphate at the 5 splice site, generating a free upstream exon, as well as a lariat carrying the intron and the downstream exon. In the sec- ond step, the 3 -OH moiety of the upstream exon attacks the 3 splice site of the Fig. 1 Representative splicing cis-elements and trans-factors. Tissue-specific and developmental stage-specific expressions of splicing trans-factors including SR proteins and hnRNP A1 enable precise regulations of alternative splicing. ISE and ISS have similar activities as ESE and ESS, but are omitted from the figure 402 K. Ohno and A. Masuda lariat leading to intron excision and ligation of the upstream and downstream exons (Query et al., 1996). In addition to the “classical” spliceosomal mechanisms, splicing is modulated by exonic/intronic splicing enhancers/silencers (ESE, ISE, ESS, ISS). The trans- factors for the splicing enhancers/silencers carry repeats of arginine and serine are accordingly called SR proteins. Tissue-specific and developmental stage-specific expressions of the splicing trans-factors enable precise spatial and temporal reg- ulations of the gene expressions. In addition, the splicing trans-factors also work on constitutively spliced exons to compensate for highly degenerative “classical” splicing cis-elements. 3 Disorders Associated with Disruption of Splicing Cis-Elements 3.1 Aberrations of the 5 Splice Sites Mutations disrupting the 5 splice sites have been most frequently reported. U1 snRNA recognizes three nucleotides at the end of an exon and six nucleotides at the beginning of an intron (Fig. 2). The completely matched nucleotides to U1 snRNA are CAG|GTAAGT, where the vertical line represents the exon/intron boundary. The completely matched sequence is observed at 1597 sites out of the entire 189,249 5 splice sites in the human genome (Sahashi et al., 2007), which is the tenth most com- mon sequence. The completely matched 5 splice site is rather avoided because, in the second stage of splicing, U1 snRNA is substituted for U5 snRNA. If U1 snRNA is tightly bound to the 5 splice site, it hinders binding of U5 snRNA. Fig. 2 U1 snRNA recognizes three nucleotides at the 3 end of an exon and six nucleotides at the 5 endofanintron Degeneracy of the 5 splice site and its vulnerability to disease-causing mutations have been extensively studied. Three algorithms have been proposed. First, Shapiro and Senapathy collated nucleotide frequencies at each position of the 5 splice site. They assumed that nucleotide frequencies at each position of the 5 splice site repre- sent the splicing signal intensity. They thus constructed a linear regression model so that the most preferred 5 splice site becomes 1.0 and the most unfavorable 5 splice site becomes 0.0 (Shapiro and Senapathy, 1987). Second, Rogan and Schneider RNA Pathologies in Neurological Disorders 403 invented the information contents, Ri. For example, at a specific position, if a single nucleotide is exclusively used, the information content at this position becomes– log 2 (1/4) = 2 bits. Similarly, if two nucleotides are equally used, the information content becomes –log 2 (2/4) = 1 bit. In Ri, the similarity to the consensus sequence is represented by the sum of information bits (Rogan and Schneider, 1995; O’Neill et al., 1998). Third, we found that a new parameter, the SD-Score, which repre- sents a common logarithm of the frequency of a specific 5 splice site in the human genome, efficiently predicts the splicing signal intensity (Sahashi et al., 2007). Our algorithm predicts the splicing consequences of mutations with the sensitiv- ity of 97.1% and the specificity of 94.7%. Simulation of all the possible mutations in the human genome using the SD-score algorithm predicts high frequencies of splicing mutations from exon –3 to intron +6 (Table 1). Especially at exon posi- tion –3, about one third of mutations are predicted to cause aberrant splicing. Using our algorithm, we predicted and proved that DYSF G1842D in Miyoshi myopathy, ABCD1 R545W in adrenoleucodystrophy, GLA Q333X in Fabry disease, and DMD Q119X and Q1144X in Duchenne muscular dystrophy are not missense or nonsense mutations but are splicing mutations. Algorithms by us and by others all point to the notion that aberrant splicing caused by mutations at the 5 splice sites is likely to be underestimated. Table 1 Predicted ratios of exonic and intronic splicing mutations Position –3 –2 –1 +1 +2 +3 +4 +5 +6 Complementary nucleotide C (%) A (%) G (%) G T A (%) A (%) G (%) T (%) A 1.8–93.7––––93.956.9 C – 89.6 99.7 – – 99.9 94.4 98.6 75.4 G 35.0 90.5 – – – 48.7 96.2 – 56.7 T 76.7 86.2 97.1 – – 99.9 94.3 97.0 – All mutations 37.8 88.8 96.8 – – 82.8 95.0 96.5 63.0 3.2 Human Branch Point Consensus Sequence In an effort to seek an algorithm to predict the position of the branch point sequence (BPS) in humans, we sequenced 367 clones of lariat RT-PCR products arising from 52 introns of 20 human housekeeping genes and identified that the human consensus BPS is simply yUnAy, where “y” represents U or C (Gao et al., 2008) (Fig. 3). The consensus BPS was more degenerative than we had expected and we failed to construct a dependable algorithm that predicts the position of the BPS. Sixteen disease-causing mutations and a polymorphism, however, have been reported to date that disrupt a BPS and cause aberrant splicing (Gao et al., 2008). Among these, eight mutates U at position –2, whereas nine affects A at position 0, which also supports the notion that U at –2 and A at 0 are essential nucleotides. 404 K. Ohno and A. Masuda Fig. 3 Human consensus BPS. (a) Pictogram and (b) WebLogo presentations of BPS. Position 0 represents the branch point. (c) Representative sequences and positions of splicing cis-elements 3.3 Ectopic AG Dinucleotide Abrogates the AG-Scanning Mechanism The 3 end of an intron and the 5 end of an exon carry a consensus sequence of CAG|G, where the vertical line represents the intron/exon boundary. The AG din- ucleotide is scanned from the branch point and the first AG is recognized as the 3 end of the intron (Chen et al., 2000). In a patient with congenital myasthenic syndrome, we identified duplication of a 16-nt segment comprised of 8 intronic and 8 exonic nucleotides at the intron 10/exon 10 boundary of CHRNE encoding the acetylcholine receptor epsilon subunit (Ohno et al., 2005). We found that the upstream AG of the duplicated segment is exclusively used for splicing and that one or two mutations in the upstream BPS had no effect whereas complete deletion of the upstream BPS partially activated the downstream AG. Similar exclusive acti- vation of the upstream AG is reported in HEXB (Dlott et al., 1990) and SLC4A1 (Bianchi et al., 1997). Creation of a cryptic AG dinucleotide close to the 3 end of an intron should be carefully scrutinized in mutation analysis. 3.4 Mutations That Disrupt ESE and ESS Gorlov and colleagues predicted that more than 16–20% of missense mutations are splicing mutations that disrupt an ESE (Gorlov et al., 2003). According to our own . 3 end of an intron to form a spliceosome complex E (Sperling et al., 2008). SF1, a 75 kDa protein, is a mammalian homologue of yeast BBP (branch point-binding protein). U2AF65 and U2AF35 bring U2. families. Invest Clin 33:13–31 Moreno-Fuenmayor H, Borjas L, Arrieta A, Valera V, Socorro-Candanoza L (1996) Plasma excitatory amino acids in autism. Invest Clin 37:113–128 Narayan M, Srinath S,. of increasing serotonergic activity during brain development: a role in autism? Int J Dev Neurosci 23:75–83 Whitaker-Azmitia PM, Lauder JM, Shemmer A, Azmitia EC (1987) Postnatal changes in serotonin receptors