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Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis a
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Theresa A Gipson , Andreas Neueder , Nancy S Wexler , Gillian P Bates & David Housman
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Koch Institute for Integrative Cancer Research; Massachusetts Institute of Technology; Cambridge, MA USA b
Department of Medical and Molecular Genetics; King’s College London; London, UK
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Hereditary Disease Foundation; New York, NY USA
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Department of Neurology and Psychiatry; Columbia University; New York, NY USA Published online: 11 Oct 2013.
To cite this article: Theresa A Gipson, Andreas Neueder, Nancy S Wexler, Gillian P Bates & David Housman (2013) Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis, RNA Biology, 10:11, 1647-1652, DOI: 10.4161/rna.26706 To link to this article: http://dx.doi.org/10.4161/rna.26706
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Theresa A Gipson1,†, Andreas Neueder2,†, Nancy S Wexler3,4, Gillian P Bates2 , and David Housman1,* 1 Koch Institute for Integrative Cancer Research; Massachusetts Institute of Technology; Cambridge, MA USA; 2Department of Medical and Molecular Genetics; King’s College London; London, UK; 3Hereditary Disease Foundation; New York, NY USA; 4Department of Neurology and Psychiatry; Columbia University; New York, NY USA † These authors contributed equally to this work.
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Keywords: HTT exon 1, missplicing, huntingtin fragment, SRSF6, Huntington’s disease *Correspondence to: David Housman; Email: [email protected] Submitted: 06/15/2013 Revised: 08/15/2013 Accepted: 10/04/2013 http://dx.doi.org/10.4161/rna.26706 Sathasivam K, Neueder A, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RL, Roos RA, Howland D, et al. Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A 2013; 110:2366-70; PMID:23341618; http://dx.doi. org/10.1073/pnas.1221891110
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untington’s disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal. The mutant huntingtin protein is processed into several smaller fragments, which have been implicated as critical factors in HD pathogenesis. The search for proteases responsible for their production has led to the identification of several cleavage sites on the huntingtin protein. However, the origin of the small N-terminal fragments that are found in HD postmortem brains has remained elusive. Recent mapping of huntingtin fragments in a mouse model demonstrated that the smallest N-terminal fragment is an exon 1 protein. This discovery spurred our hypothesis that mis-splicing as opposed to proteolysis could be generating the smallest huntingtin fragment. We demonstrated that mis-splicing of mutant huntingtin intron 1 does indeed occur and results in a short polyadenylated mRNA, which is translated into an exon 1 protein. The exon 1 protein fragment is highly pathogenic. Transgenic mouse models containing just human huntingtin exon 1 develop a rapid onset of HD-like symptoms. Our finding that a small, mis-spliced HTT transcript and corresponding exon 1 protein are
produced in the context of an expanded CAG repeat has unraveled a new molecular mechanism in HD pathogenesis. Here we present detailed models of how mis-splicing could be facilitated, what challenges remain in this model, and implications for therapeutic studies. Background Huntington’s disease (HD) is caused by a pathogenic CAG repeat expansion located in the first exon of the human huntingtin gene (HTT), and is fully penetrant when an individual has (CAG) ≥ 40.1 The HTT gene encodes huntingtin (HTT), a large ~350 kDa scaffolding protein involved in vesicle transport and transcriptional regulation (reviewed in ref. 2). The CAG triplet codes for the amino acid glutamine. An expansion of the glutamine tract causes HTT to adopt abnormal interactions and disrupt many cellular processes. An array of mouse models has been developed to help tease apart the consequences of mutant HTT and test potential therapeutics. The mouse homolog of HTT shares 91% identity at the amino acid level with the human protein (NCBIBLAST; Accessions: NP_034544.1 and NP_002102.4).3 Mouse HTT contains a repeat of seven glutamines in exon 1. This repeat, however, is encoded by the sequence (CAG) 2 (CAA)(CAG) 4, which is not prone to expansion. Mouse models of HD are either transgenic for a mutant version of human HTT or an
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Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis
Huntingtin Exon 1-Intron 1 Junction N-terminal fragment of HTT, or have had an expanded CAG repeat knocked into the endogenous mouse Htt gene. Very small N-terminal fragments have been isolated from detergent insoluble aggregates found in postmortem HD human brains.4 A comparison of the N-terminal fragments generated from wild-type and mutant HTT in the HdhQ150 knockin model of HD found that the very small HTT fragments were only derived from the mutant and not wild-type proteins.5 The smallest of these N-terminal fragments was an exon 1 HTT protein, known to be highly pathogenic when expressed in transgenic mice.5 We investigated the hypothesis that this fragment in the HdhQ150 brain is created by mis-splicing and not proteolysis by examining the exon 1-intron 1 junction of Htt mRNA.6
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The major eukaryotic 5′-splice site consensus sequence is MAG|GURAGU (M is A or C and R is A or G; -3 to position +6 relative to the exon–intron junction).7 The huntingtin 5′-splice site of intron 1, identical in mouse and human, has cytosines at positions -2 and -1, which is rare.7 Despite this unusual sequence composition, this splice site is predicted to have a rather high splice site strength based on maximum entropy modeling (Fig. 1A).8 Clustering of G and C repeats in the adjacent downstream intronic sequence probably helps to increase splice site strength, as has been demonstrated for other introns.9 In fact, mouse Htt and human HTT have > 75% GC content in the first 55 bases downstream of the intron 1 5′-splice site. Similarly upstream, between the CAG repeat
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Figure 1. Architecture of the 5′-splice site for HTT Intron 1. (A) The exon 1-intron 1 junction is conserved between human and mouse and predicted to be a strong splice site. There is an in-frame stop codon within the first four bases of intron 1. 5′-splice site consensus sequences for high GC isochores showing nucleotide conservation at the respective positions (plotted with data from ref. 7). Cytosines in the -1 and -2 positions, as for HTT exon 1-intron 1 splice site, are rare. (B) The 3′ end of HTT exon 1 is underrepresented in next generation sequencing. Read coverage is shown for the HTT 5′UTR through the beginning of intron 1. Coverage is very shallow across the 3′ end of exon 1. Histograms of read coverage were created across the HTT gene. Reads were tabulated for 150 base stretches. Coverage follows a normal distribution with the 150 base stretch at the end of exon 1/ beginning of intron 1 being consistently one of the lowest regions.
and 5′-splice site, there is a tract of > 80% GC bases in mouse Htt and human HTT. The combination of the secondary structure adopted by the CAG repeat10 and the almost 200 bases of extremely high GC content present significant challenges for sequence analysis of the exon 1-intron 1 boundary region. This challenge was so intense that this region of mouse Htt proves to be a “dead zone” in most RNA-Seq data. This was not an artifact of the RNA-Seq preparation, but is evident in other forms of next generation sequencing data. For example, we show here whole genome sequencing data from three individuals affected with HD derived from a study performed in the course of a search for modifiers of age of onset of HD by the Hereditary Disease Foundation (HDF). We show here the extremely poor read coverage at the 3′ end of HTT exon 1 (Fig. 1B) in these individuals. The HTT exon 1-intron 1 region was consistently one of the lowest covered regions of the gene. We systematically evaluated the possible ways in which the sequence coverage could be improved for this region of the huntingtin gene. For our RNA-Sequencing, we found that addition of betaine in the PCR amplification step of the RNASeq protocol helped reduce secondary structure in the region, improving overall read coverage, and the use of the highly processive Kappa polymerase improved sequence fidelity through this GC-rich region. We note that the issues of high GC content and secondary structure are also significant concerns in other genomic regions and may be of particular concern in analysis of exon1-intron1 boundaries for both RNA-Seq and whole genome sequencing for other genes as well. The sequence at the exon 1-intron 1 boundary completes the codon for a proline residue and then terminates in a stop codon. Intron 1 is extremely long and has several predicted cryptic polyA cleavage sites (Softberry POLYAH algorithm; linux1.softberry.com/all.htm). Generally, these intronic polyA sites are protected by U1 snRNP such that they cannot be recognized by cleavage and polyadenylation factors (CPSF) associated with RNA Pol II.11 If these intronic polyA cleavage sites were recognized, either because splicing
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Figure 2. Diagram of the huntingtin transcript producing an exon 1 protein. The expanded repeat is represented by hash marks in exon 1, the stop codon as a red polygon, and polyA signal with a star. We observed exon 1-intron 1 transcripts for mouse and human mutant huntingtin that corresponded with cleavage at polyA signals in intron 1. Because of the immediate stop codon, the short transcript is translated into an exon 1 protein.
and through RNA co-immunoprecipitation we demonstrated preferential association of SRSF6 with mutant as compared with wild-type Htt RNA.6 SRSF6 (SRp55) is a member of the serine/arginine (SR) family of splicing factors. These proteins share a similar structure: one or two RNA recognition motifs (RRM) and a serineand arginine-rich (SR) domain (Fig. 3A). SR proteins modulate splice site selection through RNA binding and interactions with U1 snRNP and other proteins of the spliceosome.13 While SR proteins typically enhance splicing, SRSF6 has demonstrated negative regulation of a 5′-splice site and can act antagonistically to another SR protein.14,15 For antagonistic actions between splicing regulators, local concentrations of the respective proteins are critical for proper splice site usage.15 Given that SRSF6 can both negatively regulate splicing and can potentially interact with U1 snRNP, there are two possible mechanisms by which it can influence huntingtin splicing: a large SRSF accumulation near the 5′-splice site may inhibit splicing of intron 1 or SRSF6 binding to the expanded CAG repeat may sequester the local U1 snRNP. Both of these would promote recognition of intronic polyA signals and the formation of short polyadenylated transcripts (Fig. 3B). Architecture of the Huntingtin Gene and its Influence on Splicing The huntingtin gene structure is a good example of some general features
that higher eukaryotic genes have acquired during the course of evolution. A number of bioinformatic surveys established that the first intron of a eukaryotic gene tends to be longer than subsequent introns16-23 and in humans and mice the first intron tends to be a little less than three times longer.16 The huntingtin gene is a dramatic example of this. Human HTT consists of 66 introns with an average intron length of ~2360 bases, while intron 1 alone is comprised of 11 850 bases. This discrepancy is even more pronounced in mice: average intron length is ~2080 bases, whereas intron 1 is 20 632 bases. In many species intron 1 length seems to be positively regulated with the expression level of the respective gene.21,24-29 Furthermore, neuronal genes and genes that are involved in development seem to have a higher content of non-coding DNA, which might assist in the tight regulation of their expression.17 In plants, the propensity of these elements to influence gene expression was termed intron-mediated enhancement.30,31 However, not much is known about the exact nature of these cis-acting regulatory elements in the first introns of higher animals. To date, there are no annotated non-coding RNAs in either human HTT or mouse Htt intron 1 (http://www.ensembl.org). There is also no evidence for a cryptic exon, which tends to appear in longer introns.32 So the question remains, does intron 1 of huntingtin have any particular cis-regulatory effects on the expression level of the transcript?
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of intron 1 was too slow or the protection by U1 was impeded, then a short polyadenylated huntingtin transcript would be created. Because of the immediate stop codon in intron 1, this small huntingtin transcript would encode an exon 1 HTT protein (Fig. 2). Using 3′ RACE, we demonstrated that a polyadenylated exon 1-intron 1 transcript is produced in the HdhQ150 mouse model.6 This transcript was cleaved at a polyA signal located approximately 680 bases into intron 1. We tested four other knock-in lines with expanded CAG repeats (from 50–190 CAGs) and a control knock-in line with a repeat length in the unaffected range (20 CAGs). The short polyadenylated transcript was detected at a level that increased with CAG repeat length in the expanded repeat lines and was absent from the control line.6 To confirm our findings by an independent method, we used RNA-Seq to “visualize” the HTT exon 1-intron 1 boundary. We were able to map a large density of reads back to the first 1.2 kb of intron 1 in HdhQ150 homozygote animals but not their wild-type littermates.6 These reads extend to a second polyA cleavage site, which has a stronger prediction signal than the first site at 680 bases. By comparing the short transcript 3′ UTR read coverage to the full-length Htt 3′ UTR read coverage using Mixture-of-Isoforms software,12 we were able to estimate that ~20% of Htt transcripts were mis-spliced.6 In addition, we found early intronic 1 sequences present in polysome fractions isolated from mutant but not wild-type animals, suggesting active translation of the mis-spliced RNA.6 After demonstrating that aberrant splicing occurred in the context of the mouse Htt gene, we performed 3′ RACE on the human gene using mice transgenic for fulllength human HTT. We were able to detect polyadenylated exon 1-intron 1 transcripts that extended just beyond a polyA cleavage site approximately 7.3 kilobases into intron 1. This same HTT transcript was detected in human HD postmortem brain.6 Our next step was to determine what splicing factors could be associating with HTT exon 1 that might trigger the missplicing. Using computational prediction of binding sites, we found that the splicing factor SRSF6 maps to the CAG repeat itself,
An extremely long intron 1 increases the potential for splice factor binding sites. Indeed, splice factor binding sites are scattered all along HTT intron 1, but cluster toward the 5′-end (SFmap33 and ESEfinder34). Additionally, transcription of longer intronic sequences not only allows for increased spatial, but also increased temporal regulation of splicing. While splicing is thought to usually occur co-transcriptionally,35 the extreme length of HTT intron 1 could open up a kinetic window for additional factors to act on transcription, splicing, and/or cryptic polyadenylation site activation. In addition, the rare splice site sequence discussed earlier might result in a reduction in the kinetics of U1 small nuclear ribonucleoprotein complex recruitment or the higher instability of spliceosomes. This could lead to a delay in splicing and could contribute to the generation of the small, non-spliced polyadenylated transcript. Another interesting layer of potential regulation is the local chromatin structure of HTT intron 1. The current methodology (chromatin immunoprecipitation and crosslinking immunoprecipitation
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in combination with high-throughput sequencing) would make it possible to study the effect of poly-glutamine repeat expansion on the change in chromatin marks or the association of chromatin remodelling machinery, both of which have been shown to influence splicing.36,37 The outcome of these experiments might bring further valuable insights into the molecular mechanism by which the HTT exon 1 transcript is produced. Implications of Mis-Spliced Product We know that an exon 1 HTT protein is highly pathogenic. Expression of HTT exon 1 in R6/2 transgenic mice, at a level less than that of the endogenous huntingtin gene, results in the most severe HD-like pathology that exists among the widely used mouse models of HD.38 To understand the extent to which the HTT exon 1 protein, produced through the mis-splicing mechanism described here, may contribute to disease, it is critical to study the fragment in the context of human HD. Exon 1 and exon 1-like HTT proteins, which originally stimulated our investigation of their origin, can
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Figure 3. Involvement of SRSF6 in HTT mis-splicing. (A) SRSF6 is a classical SR protein with two RNA recognition motifs (RRM) and a serine and arginine rich (SR) domain. The SRSF6 binding motif resembles a CAG repeat. (B) We propose that SRSF6 binds to the expanded CAG repeat (hash marks). Two possible scenarios could arise from this: SRSF6 binding could interfere with U1 snRNP protection of the cryptic polyA signals in intron 1 by depleting the local pool of U1 snRNP by direct interaction; or SRSF6 binding interferes with the assembly of a stable and productive spliceosome at the 5′-splice site.
be found both in knock-in mouse models5 and in postmortem HD human brain.4,39 A detailed and quantitative investigation of the relationship between the presence of these fragments and the mis-splicing of the human HTT gene is now essential. Given the extreme pathogenicity of the exon 1 HTT protein, we would expect that the frequency of mis-splicing must occur at a very low level in adult-onset HD. Our mouse data indicate that repeat size plays a critical role in frequency of mis-splicing, consistent with our working model in which the length of the repeat dictates the association of SRSF6 and exon 1. Indeed, although a low level of mis-splicing was found in the knockin line carrying 50 CAGs, which would be at the higher end of the repeat length present in most adult-onset HD patients, we were not able to detect the corresponding protein by immunoprecipitation and western blotting. In contrast, the exon 1 proteins produced via mis-splicing events in knock-in mice carrying between 80 and 190 glutamines were readily identifiable.6 This discrepancy is likely to be a combination of both a low level of mis-splicing in the 50Q mice and the polyQ lengthrelated binding kinetics of polyQ-specific antibodies. Technical considerations for identifying the mis-spliced products in human tissues can be predicted to be yet more challenging. Thus far, we have been able to detect the presence of the short mRNA by 3′RACE in postmortem brain from three HD individuals, two juvenile cases each with an expanded CAG repeat of 72 and an adult onset case with 42 CAGs (9 h postmortem delay).6 We were unsuccessful in another postmortem brain sample, possibly due to the poor quality of the RNA, as in this case the postmortem delay was 16 h. The successful 3′RACE experiments support the prediction that the misspliced HTT transcripts in human HD brains are approximately 7300 bp, which adds another level of difficulty, as the isolation of polyadenylated transcripts of this length from postmortem brain tissue is likely to be extremely difficult. This, in combination with the GC-rich sequence in this region has meant that we have not been able to show increased levels of intron 1 transcripts in RNA extracted from HD
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Figure 4. Human RNA-Seq data from Allen Brain Atlas demonstrates low representation of exon 1 coverage. RNA-Seq data from Allen is available as a table of exonic RPKMs. Plotted are HTT RPKMs for three individuals in four brain regions: cerebellar cortex (CBC), primary motor cortex (M1C), primary somatosensory cortex (S1C), and striatum (STR). RPKMs were averaged for all HTT exons (black) compared with RPKMs for exon 1 alone (red).
is targeting HTT RNA for degradation using either antisense oligonucleotides or RNAi technologies.40-42 Therapies targeting HTT RNA downstream of exon 1 will only reduce levels of a full-length HTT transcript leaving the exon 1 misspliced transcript untouched. We suggest an optimal strategy for HD therapeutics will target the RNA at the 5′ UTR or in exon 1 in order to reduce levels of both the full-length and the short mis-spliced exon 1 HTT transcript. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
The authors wish to dedicate this paper to the memory of Officer Sean Collier for his caring service to the MIT community and for his sacrifice. The investigation of HTT mis-splicing was supported by Grant G0801314 from the Medical Research Council, a grant from the CHDI Foundation, and the Koch Institute Support (core) Grant P30-CA14051from the National Cancer Institute. The whole genome sequencing studies on individuals affected with HD were performed collaboratively with Dr Wexler and were supported by the Hereditary Disease Foundation and funding from the Hearst Foundation, Grant
#GR-000014002. We are indebted to the Venezuelan families whose generous collaboration is critical to the success of many projects. References Bates G, Harper PS, Jones L. Huntington’s Disease. Oxford University Press; 2002. 2. Cattaneo E, Zuccato C, Tartari M. Normal huntingtin function: an alternative approach to Huntington’s disease. Nat Rev Neurosci 2005; 6:91930; PMID:16288298; http://dx.doi.org/10.1038/ nrn1806 3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403-10; PMID:2231712; http://dx.doi. org/10.1016/S0022-2836(05)80360-2 4. Lunkes A, Lindenberg KS, Ben-Haïem L, Weber C, Devys D, Landwehrmeyer GB, Mandel JL, Trottier Y. Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 2002; 10:25969; PMID:12191472; http://dx.doi.org/10.1016/ S1097-2765(02)00602-0 5. Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, Finkbeiner S, Sun B, Gafni J, Ellerby LM, Trottier Y, et al. Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 2010; 285:880823; PMID:20086007; http://dx.doi.org/10.1074/jbc. M109.075028 6. Sathasivam K, Neueder A, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RL, Roos RA, Howland D, et al. Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A 2013; 110:2366-70; PMID:23341618; http://dx.doi. org/10.1073/pnas.1221891110 7. Zhang MQ. Statistical features of human exons and their flanking regions. Hum Mol Genet 1998; 7:91932; PMID:9536098; http://dx.doi.org/10.1093/ hmg/7.5.919 1.
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postmortem brain and have only shown this convincingly in RNA from juvenile HD fibroblast cultures.6 Human RNA-Seq data from postmortem samples suffers from the same sequencing difficulties as noted previously, but also extreme 5′end loss of coverage. Poor RNA quality from postmortem brains means reads from the 5′ end of very long transcripts are largely lost. To illustrate this bias, we show previous human brain RNA-Seq data available in the BrainSpan atlas of the Allen Institute for Brain Science (http://www.brainspan.org/ static/home). Single-end, polyA-selected RNA-Seq was performed on different brain tissue from many individuals and made available as “reads per kilobase of exon per million mapped reads” (RPKMs) for every exon. We selected three individuals and four brain regions: cerebellar cortex (CBC), primary motor cortex (M1C), primary somatosensory cortex (S1C), and striatum (STR). We plotted the comparison between average RPKMs for all HTT exons vs. exon 1 to demonstrate how few reads are present at the 5′ end of HTT (Fig. 4). In order to quantify the level of missplicing at the exon1-intron 1 boundary, we now plan to use ribosome protection assays followed by deep sequencing to detect ribosome-protected RNA fragments. This approach will give a clean look at all the brain transcripts that were being actively translated, permitting us to determine the level at which mis-splicing occurs in human HD brain. The pathogenic contribution of the exon 1 protein produced would be expected to be a factor of both the frequency of the mis-splicing event and the half-life of the protein fragment. Even if frequency of the missplicing event is comparatively low in the adult-onset HD brain, the accumulation of a highly pathogenic protein species that is resistant to degradation would still be expected to have a strong pathogenic impact. The demonstration that the production of the HTT exon 1 occurs at a pathologically relevant scale in human brain will be directly relevant to therapeutic strategies now under development for HD. One major approach currently under development in HD therapeutics
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19. Kalari KRK, Casavant MM, Bair TBT, Keen HL, Comeron JM, Casavant TL, Scheetz TE. First exons and introns--a survey of GC content and gene structure in the human genome. In Silico Biol 2006; 6:237-42; PMID:16922687 20. Kriventseva EV, Gelfand MS. Statistical analysis of the exon-intron structure of higher and lower eukaryote genes. J Biomol Struct Dyn 1999; 17:281-8; PMID:10563578; http://dx.doi.org/10.1080/073911 02.1999.10508361 21. Marais G, Nouvellet P, Keightley PD, Charlesworth B. Intron size and exon evolution in Drosophila. Genetics 2005; 170:481-5; PMID:15781704; http:// dx.doi.org/10.1534/genetics.104.037333 22. Smith MW. Structure of vertebrate genes: a statistical analysis implicating selection. J Mol Evol 1988; 27:45-55; PMID:3133487; http://dx.doi. org/10.1007/BF02099729 23. Zhang Q, Edwards SV. The evolution of intron size in amniotes: a role for powered flight? Genome Biol Evol 2012; 4:1033-43; PMID:22930760; http://dx.doi. org/10.1093/gbe/evs070 24. Jonsson JJ, Foresman MD, Wilson N, McIvor RS. Intron requirement for expression of the human purine nucleoside phosphorylase gene. Nucleic Acids Res 1992; 20:3191-8; PMID:1620616; http://dx.doi. org/10.1093/nar/20.12.3191 25. Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster RL. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci U S A 1991; 88:478-82; PMID:1988947; http:// dx.doi.org/10.1073/pnas.88.2.478 26. Rose AB, Last RL. Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1. Plant J 1997; 11:455-64; PMID:9107035; http://dx.doi. org/10.1046/j.1365-313X.1997.11030455.x 27. Ho SH, So GMK, Chow KL. Postembryonic expression of Caenorhabditis elegans mab-21 and its requirement in sensory ray differentiation. Dev Dyn 2001; 221:422-30; PMID:11500979; http://dx.doi. org/10.1002/dvdy.1161 28. Jeon JS, Lee S, Jung KH, Jun SH, Kim C, An G. Tissue-preferential expression of a rice alpha-tubulin gene, OsTubA1, mediated by the first intron. Plant Physiol 2000; 123:1005-14; PMID:10889249; http://dx.doi.org/10.1104/pp.123.3.1005 29. Morello LL, Bardini MM, Sala FF, Breviario DD. A long leader intron of the Ostub16 rice beta-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro. Plant J 2002; 29:33-44; PMID:12060225; http://dx.doi.org/10.1046/j.0960-7412.2001.01192.x 30. Mascarenhas DD, Mettler IJI, Pierce DAD, Lowe HWH. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol 1990; 15:913-20; PMID:2103480; http://dx.doi. org/10.1007/BF00039430
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31. Rose ABA. Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA 2002; 8:1444-53; PMID:12458797; http://dx.doi. org/10.1017/S1355838202020551 32. Roy M, Kim N, Xing Y, Lee C. The effect of intron length on exon creation ratios during the evolution of mammalian genomes. RNA 2008; 14:226173; PMID:18796579; http://dx.doi.org/10.1261/ rna.1024908 33. Akerman M, David-Eden H, Pinter RY, MandelGutfreund Y. A computational approach for genomewide mapping of splicing factor binding sites. Genome Biol 2009; 10:R30; PMID:19296853; http://dx.doi. org/10.1186/gb-2009-10-3-r30 34. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 2003; 31:3568-71; PMID:12824367; http://dx.doi.org/10.1093/nar/ gkg616 35. Han J, Xiong J, Wang D, Fu X-D. Pre-mRNA splicing: where and when in the nucleus. Trends Cell Biol 2011; 21:336-43; PMID:21514162; http://dx.doi. org/10.1016/j.tcb.2011.03.003 36. Batsché E, Yaniv M, Muchardt C. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat Struct Mol Biol 2006; 13:229; PMID:16341228; http://dx.doi.org/10.1038/ nsmb1030 37. Luco RF, Pan Q, Tominaga K, Blencowe BJ, PereiraSmith OM, Misteli T. Regulation of alternative splicing by histone modifications. Science 2010; 327:996-1000; PMID:20133523; http://dx.doi. org/10.1126/science.1184208 38. Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW, et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 1996; 87:493-506; PMID:8898202; http://dx.doi.org/10.1016/S0092-8674(00)81369-0 39. DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 1997; 277:19903; PMID:9302293; http://dx.doi.org/10.1126/ science.277.5334.1990 40. Lu X-H, Yang XW. “Huntingtin holiday”: progress toward an antisense therapy for Huntington’s disease. Neuron 2012; 74:964-6; PMID:22726826; http:// dx.doi.org/10.1016/j.neuron.2012.06.001 41. Harper SQ. Progress and challenges in RNA interference therapy for Huntington disease. Arch Neurol 2009; 66:933-8; PMID:19667213; http://dx.doi. org/10.1001/archneurol.2009.180 42. Sah DWY, Aronin N. Oligonucleotide therapeutic approaches for Huntington disease. J Clin Invest 2011; 121:500-7; PMID:21285523; http://dx.doi. org/10.1172/JCI45130
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8. Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 2004; 11:377-94; PMID:15285897; http://dx.doi. org/10.1089/1066527041410418 9. Xiao X, Wang Z, Jang M, Nutiu R, Wang ET, Burge CB. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat Struct Mol Biol 2009; 16:1094-100; PMID:19749754; http://dx.doi. org/10.1038/nsmb.1661 10. de Mezer M, Wojciechowska M, Napierala M, Sobczak K, Krzyzosiak WJ. Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference. Nucleic Acids Res 2011; 39:3852-63; PMID:21247881; http://dx.doi.org/10.1093/nar/gkq1323 11. Berg MG, Singh LN, Younis I, Liu Q, Pinto AM, Kaida D, Zhang Z, Cho S, Sherrill-Mix S, Wan L, et al. U1 snRNP determines mRNA length and regulates isoform expression. Cell 2012; 150:5364; PMID:22770214; http://dx.doi.org/10.1016/j. cell.2012.05.029 12. Katz Y, Wang ET, Airoldi EM, Burge CB. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods 2010; 7:1009-15; PMID:21057496; http://dx.doi. org/10.1038/nmeth.1528 13. Long JC, Caceres JF. The SR protein family of splicing factors: master regulators of gene expression. Biochem J 2009; 417:15-27; PMID:19061484; http://dx.doi.org/10.1042/BJ20081501 14. Tranell A, Fenyö EM, Schwartz S. Serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) induce production of HIV-1 vpr mRNA by inhibiting the 5′-splice site of exon 3. J Biol Chem 2010; 285:31537-47; PMID:20685659; http://dx.doi. org/10.1074/jbc.M109.077453 15. Labourier EE, Bourbon HMH, Gallouzi IEI, Fostier MM, Allemand EE, Tazi JJ. Antagonism between RSF1 and SR proteins for both splicesite recognition in vitro and Drosophila development. Genes Dev 1999; 13:740-53; http:// pubget.com /paper/10 090730 ?institution = mit. edu; PMID:10090730; http://dx.doi.org/10.1101/ gad.13.6.740. 16. Bradnam KR, Korf I. Longer first introns are a general property of eukaryotic gene structure. PLoS One 2008; 3:e3093; PMID:18769727; http://dx.doi. org/10.1371/journal.pone.0003093 17. Gaffney DJ, Keightley PD. Genomic selective constraints in murid noncoding DNA. PLoS Genet 2006; 2:e204; PMID:17166057; http://dx.doi. org/10.1371/journal.pgen.0020204 18. Gazave E, Marqués-Bonet T, Fernando O, Charlesworth B, Navarro A. Patterns and rates of intron divergence between humans and chimpanzees. Genome Biol 2007; 8:R21; PMID:17309804; http:// dx.doi.org/10.1186/gb-2007-8-2-r21
RNA Biology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/krnb20
Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis a
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Theresa A Gipson , Andreas Neueder , Nancy S Wexler , Gillian P Bates & David Housman
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a
Koch Institute for Integrative Cancer Research; Massachusetts Institute of Technology; Cambridge, MA USA b
Department of Medical and Molecular Genetics; King’s College London; London, UK
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Hereditary Disease Foundation; New York, NY USA
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Department of Neurology and Psychiatry; Columbia University; New York, NY USA Published online: 11 Oct 2013.
To cite this article: Theresa A Gipson, Andreas Neueder, Nancy S Wexler, Gillian P Bates & David Housman (2013) Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis, RNA Biology, 10:11, 1647-1652, DOI: 10.4161/rna.26706 To link to this article: http://dx.doi.org/10.4161/rna.26706
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Theresa A Gipson1,†, Andreas Neueder2,†, Nancy S Wexler3,4, Gillian P Bates2 , and David Housman1,* 1 Koch Institute for Integrative Cancer Research; Massachusetts Institute of Technology; Cambridge, MA USA; 2Department of Medical and Molecular Genetics; King’s College London; London, UK; 3Hereditary Disease Foundation; New York, NY USA; 4Department of Neurology and Psychiatry; Columbia University; New York, NY USA † These authors contributed equally to this work.
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Keywords: HTT exon 1, missplicing, huntingtin fragment, SRSF6, Huntington’s disease *Correspondence to: David Housman; Email: [email protected] Submitted: 06/15/2013 Revised: 08/15/2013 Accepted: 10/04/2013 http://dx.doi.org/10.4161/rna.26706 Sathasivam K, Neueder A, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RL, Roos RA, Howland D, et al. Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A 2013; 110:2366-70; PMID:23341618; http://dx.doi. org/10.1073/pnas.1221891110
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untington’s disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal. The mutant huntingtin protein is processed into several smaller fragments, which have been implicated as critical factors in HD pathogenesis. The search for proteases responsible for their production has led to the identification of several cleavage sites on the huntingtin protein. However, the origin of the small N-terminal fragments that are found in HD postmortem brains has remained elusive. Recent mapping of huntingtin fragments in a mouse model demonstrated that the smallest N-terminal fragment is an exon 1 protein. This discovery spurred our hypothesis that mis-splicing as opposed to proteolysis could be generating the smallest huntingtin fragment. We demonstrated that mis-splicing of mutant huntingtin intron 1 does indeed occur and results in a short polyadenylated mRNA, which is translated into an exon 1 protein. The exon 1 protein fragment is highly pathogenic. Transgenic mouse models containing just human huntingtin exon 1 develop a rapid onset of HD-like symptoms. Our finding that a small, mis-spliced HTT transcript and corresponding exon 1 protein are
produced in the context of an expanded CAG repeat has unraveled a new molecular mechanism in HD pathogenesis. Here we present detailed models of how mis-splicing could be facilitated, what challenges remain in this model, and implications for therapeutic studies. Background Huntington’s disease (HD) is caused by a pathogenic CAG repeat expansion located in the first exon of the human huntingtin gene (HTT), and is fully penetrant when an individual has (CAG) ≥ 40.1 The HTT gene encodes huntingtin (HTT), a large ~350 kDa scaffolding protein involved in vesicle transport and transcriptional regulation (reviewed in ref. 2). The CAG triplet codes for the amino acid glutamine. An expansion of the glutamine tract causes HTT to adopt abnormal interactions and disrupt many cellular processes. An array of mouse models has been developed to help tease apart the consequences of mutant HTT and test potential therapeutics. The mouse homolog of HTT shares 91% identity at the amino acid level with the human protein (NCBIBLAST; Accessions: NP_034544.1 and NP_002102.4).3 Mouse HTT contains a repeat of seven glutamines in exon 1. This repeat, however, is encoded by the sequence (CAG) 2 (CAA)(CAG) 4, which is not prone to expansion. Mouse models of HD are either transgenic for a mutant version of human HTT or an
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Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis
Huntingtin Exon 1-Intron 1 Junction N-terminal fragment of HTT, or have had an expanded CAG repeat knocked into the endogenous mouse Htt gene. Very small N-terminal fragments have been isolated from detergent insoluble aggregates found in postmortem HD human brains.4 A comparison of the N-terminal fragments generated from wild-type and mutant HTT in the HdhQ150 knockin model of HD found that the very small HTT fragments were only derived from the mutant and not wild-type proteins.5 The smallest of these N-terminal fragments was an exon 1 HTT protein, known to be highly pathogenic when expressed in transgenic mice.5 We investigated the hypothesis that this fragment in the HdhQ150 brain is created by mis-splicing and not proteolysis by examining the exon 1-intron 1 junction of Htt mRNA.6
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The major eukaryotic 5′-splice site consensus sequence is MAG|GURAGU (M is A or C and R is A or G; -3 to position +6 relative to the exon–intron junction).7 The huntingtin 5′-splice site of intron 1, identical in mouse and human, has cytosines at positions -2 and -1, which is rare.7 Despite this unusual sequence composition, this splice site is predicted to have a rather high splice site strength based on maximum entropy modeling (Fig. 1A).8 Clustering of G and C repeats in the adjacent downstream intronic sequence probably helps to increase splice site strength, as has been demonstrated for other introns.9 In fact, mouse Htt and human HTT have > 75% GC content in the first 55 bases downstream of the intron 1 5′-splice site. Similarly upstream, between the CAG repeat
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Figure 1. Architecture of the 5′-splice site for HTT Intron 1. (A) The exon 1-intron 1 junction is conserved between human and mouse and predicted to be a strong splice site. There is an in-frame stop codon within the first four bases of intron 1. 5′-splice site consensus sequences for high GC isochores showing nucleotide conservation at the respective positions (plotted with data from ref. 7). Cytosines in the -1 and -2 positions, as for HTT exon 1-intron 1 splice site, are rare. (B) The 3′ end of HTT exon 1 is underrepresented in next generation sequencing. Read coverage is shown for the HTT 5′UTR through the beginning of intron 1. Coverage is very shallow across the 3′ end of exon 1. Histograms of read coverage were created across the HTT gene. Reads were tabulated for 150 base stretches. Coverage follows a normal distribution with the 150 base stretch at the end of exon 1/ beginning of intron 1 being consistently one of the lowest regions.
and 5′-splice site, there is a tract of > 80% GC bases in mouse Htt and human HTT. The combination of the secondary structure adopted by the CAG repeat10 and the almost 200 bases of extremely high GC content present significant challenges for sequence analysis of the exon 1-intron 1 boundary region. This challenge was so intense that this region of mouse Htt proves to be a “dead zone” in most RNA-Seq data. This was not an artifact of the RNA-Seq preparation, but is evident in other forms of next generation sequencing data. For example, we show here whole genome sequencing data from three individuals affected with HD derived from a study performed in the course of a search for modifiers of age of onset of HD by the Hereditary Disease Foundation (HDF). We show here the extremely poor read coverage at the 3′ end of HTT exon 1 (Fig. 1B) in these individuals. The HTT exon 1-intron 1 region was consistently one of the lowest covered regions of the gene. We systematically evaluated the possible ways in which the sequence coverage could be improved for this region of the huntingtin gene. For our RNA-Sequencing, we found that addition of betaine in the PCR amplification step of the RNASeq protocol helped reduce secondary structure in the region, improving overall read coverage, and the use of the highly processive Kappa polymerase improved sequence fidelity through this GC-rich region. We note that the issues of high GC content and secondary structure are also significant concerns in other genomic regions and may be of particular concern in analysis of exon1-intron1 boundaries for both RNA-Seq and whole genome sequencing for other genes as well. The sequence at the exon 1-intron 1 boundary completes the codon for a proline residue and then terminates in a stop codon. Intron 1 is extremely long and has several predicted cryptic polyA cleavage sites (Softberry POLYAH algorithm; linux1.softberry.com/all.htm). Generally, these intronic polyA sites are protected by U1 snRNP such that they cannot be recognized by cleavage and polyadenylation factors (CPSF) associated with RNA Pol II.11 If these intronic polyA cleavage sites were recognized, either because splicing
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Figure 2. Diagram of the huntingtin transcript producing an exon 1 protein. The expanded repeat is represented by hash marks in exon 1, the stop codon as a red polygon, and polyA signal with a star. We observed exon 1-intron 1 transcripts for mouse and human mutant huntingtin that corresponded with cleavage at polyA signals in intron 1. Because of the immediate stop codon, the short transcript is translated into an exon 1 protein.
and through RNA co-immunoprecipitation we demonstrated preferential association of SRSF6 with mutant as compared with wild-type Htt RNA.6 SRSF6 (SRp55) is a member of the serine/arginine (SR) family of splicing factors. These proteins share a similar structure: one or two RNA recognition motifs (RRM) and a serineand arginine-rich (SR) domain (Fig. 3A). SR proteins modulate splice site selection through RNA binding and interactions with U1 snRNP and other proteins of the spliceosome.13 While SR proteins typically enhance splicing, SRSF6 has demonstrated negative regulation of a 5′-splice site and can act antagonistically to another SR protein.14,15 For antagonistic actions between splicing regulators, local concentrations of the respective proteins are critical for proper splice site usage.15 Given that SRSF6 can both negatively regulate splicing and can potentially interact with U1 snRNP, there are two possible mechanisms by which it can influence huntingtin splicing: a large SRSF accumulation near the 5′-splice site may inhibit splicing of intron 1 or SRSF6 binding to the expanded CAG repeat may sequester the local U1 snRNP. Both of these would promote recognition of intronic polyA signals and the formation of short polyadenylated transcripts (Fig. 3B). Architecture of the Huntingtin Gene and its Influence on Splicing The huntingtin gene structure is a good example of some general features
that higher eukaryotic genes have acquired during the course of evolution. A number of bioinformatic surveys established that the first intron of a eukaryotic gene tends to be longer than subsequent introns16-23 and in humans and mice the first intron tends to be a little less than three times longer.16 The huntingtin gene is a dramatic example of this. Human HTT consists of 66 introns with an average intron length of ~2360 bases, while intron 1 alone is comprised of 11 850 bases. This discrepancy is even more pronounced in mice: average intron length is ~2080 bases, whereas intron 1 is 20 632 bases. In many species intron 1 length seems to be positively regulated with the expression level of the respective gene.21,24-29 Furthermore, neuronal genes and genes that are involved in development seem to have a higher content of non-coding DNA, which might assist in the tight regulation of their expression.17 In plants, the propensity of these elements to influence gene expression was termed intron-mediated enhancement.30,31 However, not much is known about the exact nature of these cis-acting regulatory elements in the first introns of higher animals. To date, there are no annotated non-coding RNAs in either human HTT or mouse Htt intron 1 (http://www.ensembl.org). There is also no evidence for a cryptic exon, which tends to appear in longer introns.32 So the question remains, does intron 1 of huntingtin have any particular cis-regulatory effects on the expression level of the transcript?
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of intron 1 was too slow or the protection by U1 was impeded, then a short polyadenylated huntingtin transcript would be created. Because of the immediate stop codon in intron 1, this small huntingtin transcript would encode an exon 1 HTT protein (Fig. 2). Using 3′ RACE, we demonstrated that a polyadenylated exon 1-intron 1 transcript is produced in the HdhQ150 mouse model.6 This transcript was cleaved at a polyA signal located approximately 680 bases into intron 1. We tested four other knock-in lines with expanded CAG repeats (from 50–190 CAGs) and a control knock-in line with a repeat length in the unaffected range (20 CAGs). The short polyadenylated transcript was detected at a level that increased with CAG repeat length in the expanded repeat lines and was absent from the control line.6 To confirm our findings by an independent method, we used RNA-Seq to “visualize” the HTT exon 1-intron 1 boundary. We were able to map a large density of reads back to the first 1.2 kb of intron 1 in HdhQ150 homozygote animals but not their wild-type littermates.6 These reads extend to a second polyA cleavage site, which has a stronger prediction signal than the first site at 680 bases. By comparing the short transcript 3′ UTR read coverage to the full-length Htt 3′ UTR read coverage using Mixture-of-Isoforms software,12 we were able to estimate that ~20% of Htt transcripts were mis-spliced.6 In addition, we found early intronic 1 sequences present in polysome fractions isolated from mutant but not wild-type animals, suggesting active translation of the mis-spliced RNA.6 After demonstrating that aberrant splicing occurred in the context of the mouse Htt gene, we performed 3′ RACE on the human gene using mice transgenic for fulllength human HTT. We were able to detect polyadenylated exon 1-intron 1 transcripts that extended just beyond a polyA cleavage site approximately 7.3 kilobases into intron 1. This same HTT transcript was detected in human HD postmortem brain.6 Our next step was to determine what splicing factors could be associating with HTT exon 1 that might trigger the missplicing. Using computational prediction of binding sites, we found that the splicing factor SRSF6 maps to the CAG repeat itself,
An extremely long intron 1 increases the potential for splice factor binding sites. Indeed, splice factor binding sites are scattered all along HTT intron 1, but cluster toward the 5′-end (SFmap33 and ESEfinder34). Additionally, transcription of longer intronic sequences not only allows for increased spatial, but also increased temporal regulation of splicing. While splicing is thought to usually occur co-transcriptionally,35 the extreme length of HTT intron 1 could open up a kinetic window for additional factors to act on transcription, splicing, and/or cryptic polyadenylation site activation. In addition, the rare splice site sequence discussed earlier might result in a reduction in the kinetics of U1 small nuclear ribonucleoprotein complex recruitment or the higher instability of spliceosomes. This could lead to a delay in splicing and could contribute to the generation of the small, non-spliced polyadenylated transcript. Another interesting layer of potential regulation is the local chromatin structure of HTT intron 1. The current methodology (chromatin immunoprecipitation and crosslinking immunoprecipitation
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in combination with high-throughput sequencing) would make it possible to study the effect of poly-glutamine repeat expansion on the change in chromatin marks or the association of chromatin remodelling machinery, both of which have been shown to influence splicing.36,37 The outcome of these experiments might bring further valuable insights into the molecular mechanism by which the HTT exon 1 transcript is produced. Implications of Mis-Spliced Product We know that an exon 1 HTT protein is highly pathogenic. Expression of HTT exon 1 in R6/2 transgenic mice, at a level less than that of the endogenous huntingtin gene, results in the most severe HD-like pathology that exists among the widely used mouse models of HD.38 To understand the extent to which the HTT exon 1 protein, produced through the mis-splicing mechanism described here, may contribute to disease, it is critical to study the fragment in the context of human HD. Exon 1 and exon 1-like HTT proteins, which originally stimulated our investigation of their origin, can
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Figure 3. Involvement of SRSF6 in HTT mis-splicing. (A) SRSF6 is a classical SR protein with two RNA recognition motifs (RRM) and a serine and arginine rich (SR) domain. The SRSF6 binding motif resembles a CAG repeat. (B) We propose that SRSF6 binds to the expanded CAG repeat (hash marks). Two possible scenarios could arise from this: SRSF6 binding could interfere with U1 snRNP protection of the cryptic polyA signals in intron 1 by depleting the local pool of U1 snRNP by direct interaction; or SRSF6 binding interferes with the assembly of a stable and productive spliceosome at the 5′-splice site.
be found both in knock-in mouse models5 and in postmortem HD human brain.4,39 A detailed and quantitative investigation of the relationship between the presence of these fragments and the mis-splicing of the human HTT gene is now essential. Given the extreme pathogenicity of the exon 1 HTT protein, we would expect that the frequency of mis-splicing must occur at a very low level in adult-onset HD. Our mouse data indicate that repeat size plays a critical role in frequency of mis-splicing, consistent with our working model in which the length of the repeat dictates the association of SRSF6 and exon 1. Indeed, although a low level of mis-splicing was found in the knockin line carrying 50 CAGs, which would be at the higher end of the repeat length present in most adult-onset HD patients, we were not able to detect the corresponding protein by immunoprecipitation and western blotting. In contrast, the exon 1 proteins produced via mis-splicing events in knock-in mice carrying between 80 and 190 glutamines were readily identifiable.6 This discrepancy is likely to be a combination of both a low level of mis-splicing in the 50Q mice and the polyQ lengthrelated binding kinetics of polyQ-specific antibodies. Technical considerations for identifying the mis-spliced products in human tissues can be predicted to be yet more challenging. Thus far, we have been able to detect the presence of the short mRNA by 3′RACE in postmortem brain from three HD individuals, two juvenile cases each with an expanded CAG repeat of 72 and an adult onset case with 42 CAGs (9 h postmortem delay).6 We were unsuccessful in another postmortem brain sample, possibly due to the poor quality of the RNA, as in this case the postmortem delay was 16 h. The successful 3′RACE experiments support the prediction that the misspliced HTT transcripts in human HD brains are approximately 7300 bp, which adds another level of difficulty, as the isolation of polyadenylated transcripts of this length from postmortem brain tissue is likely to be extremely difficult. This, in combination with the GC-rich sequence in this region has meant that we have not been able to show increased levels of intron 1 transcripts in RNA extracted from HD
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Figure 4. Human RNA-Seq data from Allen Brain Atlas demonstrates low representation of exon 1 coverage. RNA-Seq data from Allen is available as a table of exonic RPKMs. Plotted are HTT RPKMs for three individuals in four brain regions: cerebellar cortex (CBC), primary motor cortex (M1C), primary somatosensory cortex (S1C), and striatum (STR). RPKMs were averaged for all HTT exons (black) compared with RPKMs for exon 1 alone (red).
is targeting HTT RNA for degradation using either antisense oligonucleotides or RNAi technologies.40-42 Therapies targeting HTT RNA downstream of exon 1 will only reduce levels of a full-length HTT transcript leaving the exon 1 misspliced transcript untouched. We suggest an optimal strategy for HD therapeutics will target the RNA at the 5′ UTR or in exon 1 in order to reduce levels of both the full-length and the short mis-spliced exon 1 HTT transcript. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
The authors wish to dedicate this paper to the memory of Officer Sean Collier for his caring service to the MIT community and for his sacrifice. The investigation of HTT mis-splicing was supported by Grant G0801314 from the Medical Research Council, a grant from the CHDI Foundation, and the Koch Institute Support (core) Grant P30-CA14051from the National Cancer Institute. The whole genome sequencing studies on individuals affected with HD were performed collaboratively with Dr Wexler and were supported by the Hereditary Disease Foundation and funding from the Hearst Foundation, Grant
#GR-000014002. We are indebted to the Venezuelan families whose generous collaboration is critical to the success of many projects. References Bates G, Harper PS, Jones L. Huntington’s Disease. Oxford University Press; 2002. 2. Cattaneo E, Zuccato C, Tartari M. Normal huntingtin function: an alternative approach to Huntington’s disease. Nat Rev Neurosci 2005; 6:91930; PMID:16288298; http://dx.doi.org/10.1038/ nrn1806 3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403-10; PMID:2231712; http://dx.doi. org/10.1016/S0022-2836(05)80360-2 4. Lunkes A, Lindenberg KS, Ben-Haïem L, Weber C, Devys D, Landwehrmeyer GB, Mandel JL, Trottier Y. Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol Cell 2002; 10:25969; PMID:12191472; http://dx.doi.org/10.1016/ S1097-2765(02)00602-0 5. Landles C, Sathasivam K, Weiss A, Woodman B, Moffitt H, Finkbeiner S, Sun B, Gafni J, Ellerby LM, Trottier Y, et al. Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem 2010; 285:880823; PMID:20086007; http://dx.doi.org/10.1074/jbc. M109.075028 6. Sathasivam K, Neueder A, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RL, Roos RA, Howland D, et al. Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci U S A 2013; 110:2366-70; PMID:23341618; http://dx.doi. org/10.1073/pnas.1221891110 7. Zhang MQ. Statistical features of human exons and their flanking regions. Hum Mol Genet 1998; 7:91932; PMID:9536098; http://dx.doi.org/10.1093/ hmg/7.5.919 1.
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postmortem brain and have only shown this convincingly in RNA from juvenile HD fibroblast cultures.6 Human RNA-Seq data from postmortem samples suffers from the same sequencing difficulties as noted previously, but also extreme 5′end loss of coverage. Poor RNA quality from postmortem brains means reads from the 5′ end of very long transcripts are largely lost. To illustrate this bias, we show previous human brain RNA-Seq data available in the BrainSpan atlas of the Allen Institute for Brain Science (http://www.brainspan.org/ static/home). Single-end, polyA-selected RNA-Seq was performed on different brain tissue from many individuals and made available as “reads per kilobase of exon per million mapped reads” (RPKMs) for every exon. We selected three individuals and four brain regions: cerebellar cortex (CBC), primary motor cortex (M1C), primary somatosensory cortex (S1C), and striatum (STR). We plotted the comparison between average RPKMs for all HTT exons vs. exon 1 to demonstrate how few reads are present at the 5′ end of HTT (Fig. 4). In order to quantify the level of missplicing at the exon1-intron 1 boundary, we now plan to use ribosome protection assays followed by deep sequencing to detect ribosome-protected RNA fragments. This approach will give a clean look at all the brain transcripts that were being actively translated, permitting us to determine the level at which mis-splicing occurs in human HD brain. The pathogenic contribution of the exon 1 protein produced would be expected to be a factor of both the frequency of the mis-splicing event and the half-life of the protein fragment. Even if frequency of the missplicing event is comparatively low in the adult-onset HD brain, the accumulation of a highly pathogenic protein species that is resistant to degradation would still be expected to have a strong pathogenic impact. The demonstration that the production of the HTT exon 1 occurs at a pathologically relevant scale in human brain will be directly relevant to therapeutic strategies now under development for HD. One major approach currently under development in HD therapeutics
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