Part II Dynamics of pre-mRNA Splicing During the Process of Transcription The process of gene transcription, which is ubiquitous in any genetic network (such as the p53-AKT network studied in Part I), is as dynamic as the genetic network it belongs to Due to the coupling of intron splicing to pre-mRNA elongation during the process of gene transcription, the processing of pre-mRNA to mature mRNA is dynamic, which in turns lead to dynamic changes in the secondary structures of the pre-mRNA during transcription In Part II (Chapters and 7), a new model to approximate pre-mRNA folding during the process of transcription is proposed to study the dynamics of dystrophin pre-mRNA secondary structures during transcription It will be shown that the dynamic changes in the secondary structures of the pre-mRNA is a critical factor in influencing the efficacy and efficiency of pharmacological molecules to remove genetic mutations in the dystrophin pre-mRNA, a potential therapeutic strategy for Duchenne muscular dystrophy 118 Chapter Selective Exclusion of Exons as a Potential Therapy for Duchenne Muscular Dystrophy Duchenne muscular dystrophy (DMD), an X-linked recessive condition, is the most common form of muscular dystrophy occurring at a cited frequency of about in 3,500 live male births (Emery, 1989) DMD is characterized by fast progressive muscle degeneration that leads to eventual premature death of DMD patients; patients experience muscle weakness from the age of years and are typically wheelchairbounded by 12 years This devastating disease is predominantly caused by the absence of functional dystrophin protein, which plays an essential role in the muscle fiber’s structure and function (Hoffman et al., 1987; Zubrzycka-Gaarn et al., 1988; Ervasti and Campbell, 1991; Koenig et al., 1988) The common cause of dystrophin protein deficiency is genetic mutations in the huge dystrophin gene causing either premature termination of translation by nonsense mutation (i.e., premature stop codon) or disruption of the open reading frame of the mRNA transcript by out-offrame insertions/deletions (Hoffman et al., 1987; Monaco et al., 1988; Koenig et al., 1989) Due to the large size of the dystrophin coding sequence (see Section 6.1), gene therapy, which uses gene delivery systems to insert a normal dystrophin sequence into the patient’s muscle tissue, are only modestly capable of transducing sufficient muscle fibers for long time-periods (Aartsma-Rus et al., 2002; van Deutekom et al., 1998) 119 Therefore, alternatives such as pharmacological therapy or gene correction have gained increasing attention One promising pharmacological therapeutic strategy is to use synthetic molecules to induce the exclusion of specific exon(s) for the removal of genetic mutations (mechanisms are described in Section 6.2) Specifically, by skipping either exons flanking out-of-frame deletions/insertions or an in-frame exon containing a nonsense mutation, the dystrophin reading frame is restored and thereby induces the synthesis of in-frame dystrophin protein; a rule-of-thumb for determining which specific exon(s) to skip is given in Appendix A-16 Albeit the corrected dystrophin protein is slightly truncated, the severity of DMD is significantly alleviated to a relatively milder form known as Becker muscular dystrophy (BMD); DMD and BMD are caused by genetic mutations of the same gene In BMD, progressive muscle degeneration is slower and is markedly less fatal than DMD; in some cases, BMD patients are diagnosed only late in life at the age of 60 years (Heald et al., 1994) The plausibility of this particular therapeutic approach has been demonstrated in cells derived from the mdx mouse model (Dunckley et al., 1998; Wilton et al., 1999; Mann et al., 2001, 2002; Lu et al., 2003) and DMD patients (Takeshima et al., 2001; van Deutekom et al., 2001; Aartsma-Rus et al., 2002, 2003, 2006; De Angelis et al., 2002; McClorey et al., 2006a), as well as in animal models (Lu et al., 2005; Alter et al., 2006; Fletcher et al., 2006; Fall et al., 2006; McClorey et al., 2006b) These encouraging results have led to the rapid progression to clinical trials (Muntoni et al., 2005; Takeshima et al., 2006) to validate that promising in vitro results can be reproducible in human muscle under in vivo conditions 120 Given that DMD patients not manifest identical mutations, personalized therapy is necessary in which specific pharmacological molecules must be customized to address the many mutations spreading across the many exons (Wilton et al., 2007) However, as these molecules are designed generally by trial-and-error means (Aartsma-Rus et al., 2005; Wilton et al., 2007), heavy expenditure in many unsuccessful wet-lab trials are incurred, which increases the cost and waiting time of therapy Therefore, a systematic and efficient methodology in designing efficacious molecules for each patient is critical but is currently unavailable due to the lack of known major factors affecting the efficiency of exon skipping As shall be emphasized in this chapter, the dynamics of co-transcriptional binding accessibility of a target site in the dystrophin pre-mRNA, which the specific pharmacological molecule must bind in order to induce selective exon skipping, is a major factor that has been overlooked 6.1 The dystrophin gene The dystrophin gene is the largest gene known in humans with a total genomic sequence length of 2,220,382 nucleotides (Roberts et al., 1993) Surprisingly, the total coding sequence of 79 exons only constitutes 11,034 nucleotides or a mere 0.5 % of its full length DNA Moreover, it requires about 16 to 24 hours to fully transcribe a dystrophin mRNA (Tennyson et al., 1995) As a result, searching for an exon in the dystrophin transcript is akin to finding a needle in a haystack, as illustrated in Figure 6-1 121 1,000,000 N u m b e r o f b a s e p a irs 100,000 10,000 1,000 100 10 1 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Exon 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 Figure 6-1A DNA sequence length of each exonic and intronic region in the dystrophin gene The sequence lengths for each of the 79 exons in dystrophin gene are plotted as black bars For each exon, both of their flanking intronic sequence lengths are shown as gray bars Note that the sequence length is on a logarithmic scale (vertical axis) The exonic regions range from to 269 nucleotides In contrast, the intronic regions range from 107 to 319,058 nucleotides 122 P e rc e n t a g e 1 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Exon 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 Figure 6-1B Percentage length of each exonic region relative to its flanking intronic region To illustrate the fact that the exonic region of the dystrophin gene is minute relative to the intronic regions, the percentage of the length of an exon relative to the total length of its flanking intronic regions is plotted Most of the exons constitute less than 1% of their intronic lengths Indeed, the highest percentage is not more than 7% 123 The dystrophin protein consists of an N-terminal domain that binds to actin filaments, a large central rod domain, and a C-terminal cysteine-rich domain that binds to dystrophin-glycoprotein complexes (Hoffman et al., 1987; Koenig et al., 1988) Notably, substantial segments of the central rod domain are not critical for its function (England et al., 1990; Heald et al., 1994; Muntoni et al., 2003) Indeed, BMD patients have been reported to lack up to 67% of the central rod domain (England et al., 1990; Winnard et al., 1993; Mirabella et al., 1998) while retaining the N- and C-terminal ends (Koenig et al., 1989) In contrast, the lack of either terminal ends are detrimental for dystrophin functions that results in DMD Hence, given the long central rod domain, many choices of exons can be skipped depending on the position of the mutations in DMD patients, of which more than 75% of DMD patients would benefit from the skipping of these exons (Aartsma-Rus et al., 2004) 6.2 Mechanism to induce exon skipping As the mechanism to induce selective exon skipping impinges on the mechanisms for which exons are distinguished from introns, a summary of the latter is first given 6.2.1 Exon recognition sites As depicted in Figure 6-2, splicing factors, each of which comprises several nuclear proteins (e.g U1, U2 and SR) identify exons by binding to important sequences in the pre-mRNA These sequences include donor (GU) and acceptor (AG) splice sites, 124 branch points (A), pyrimidine tracts (YYYY) and exon splicing enhancers (ESEs) (Blencowe, 2000) Note that ESE sites are resided within an exon (with typically to nucleotides) whereas the rest of the exon recognition sites are located at the intronic regions These splicing factors then form the 60S splicing machinery called the spliceosome (Staley and Guthrie, 1998), which removes the introns while retaining the exons during pre-mRNA processing Generally, donor and acceptor splice sites are less specific among exons than ESE sites and furthermore, pseudo versions are prevalent throughout the genome (Alberts et al., 2000) Indeed, studies have shown that through SR (serine-rich) protein binding to specific ESE sites (Figure 6-2), accuracy of the spliceosome to identify exons is enhanced and pseudo splice sites are bypassed (Lam and Hertel, 2002; Graveley, 2000; Graveley et al., 1999; Alberts et al., 2000; Ibrahim et al., 2005) Because of the very long intronic sequences and large number of exons in the dystrophin gene (Figure 6-1), ESEs are expected to play important roles in identifying exons accurately (Lam and Hertel, 2002; Graveley, 2000; Graveley et al., 1999; Wang et al., 2005; Cartegni et al., 2002; Blencowe, 2000) Figure 6-2 Splicing factors and the respective pre-mRNA sequences to which they bind The family of SR proteins binds to ESE sites, which reside within an exon Other splicing factors such as U1 and U2 bind to exon recognition sites located at intronic sequences These splicing factors form the spliceosome that removes the introns during splicing of pre-mRNA This figure is reproduced from Nature (2002) 418:236 with permission from Nature Publishing Group 125 6.2.2 Blocking of exon recognition sites by AONs AON X–1 X X+1 Figure 6-3 Antisense oligonucleotides induce exon skipping by binding to ESE sites Schematic diagram illustrating for the case in which AON competitively binds to ESE sites against splicing factors, i.e., SR proteins Legend: exons are shown as gray rectangular boxes and ESE sites are demarcated as black regions within the exons Therefore, by blocking specific exon recognition sites from the spliceosome, recognition of the targeted exon is bypassed and is thereby removed along with the introns Antisense oligonucleotides (AONs), which are synthetic single-stranded molecules, typically consisting of 16 to 30 nucleotides that are complementary to a specific sequence in the target RNA, can be used to bind and thereby block these exon recognition sites Specifically, an AON induces exon skipping by competitive binding at its target site against splicing factors during transcription (Wilton and Fletcher, 2005b; Pramono et al., 1996), as illustrated in Figure 6-3 Apart from their well-documented applications to suppress gene expression, AONs have been used to modulate pre-messenger RNA (pre-mRNA) splicing as potential therapeutic strategy for genetic diseases such as DMD (Pramono et al., 1996; Matsuo, 1996; Matsuo and Takeshima, 2005; Wilton and Fletcher, 2005a, 2005b; Wilton et al., 2007; AartsmaRus et al., 2002, 2004, 2005, 2006; van Deutekom and van Ommen, 2003; Surono et al., 2004), thalassemia (Suwanmanee et al., 2002; Gorman et al., 2000; Sazani and Kole, 2003; Dominski and Kole, 1993), ocular albinism (Vetrini et al., 2006) and cancer (Mercatante et al., 2001) 126 Studies that target AONs to ESE sites show specific and effective induction of exon skipping in dystrophin mRNA (Matsuo, 1996; Pramono et al., 1996; AartsmaRus et al., 2006, 2004, 2002, 2005; Surono et al., 2004) but resulted in unpredictable skipping of adjacent exons when targeted to the splice sites (Aartsma-Rus et al., 2002; Mann et al., 2001; Wilton et al., 1999) In view of the importance of specificity in exon skipping, only AONs targeting ESE sites are studied 6.3 An extensive network of coupling among gene expression machines Recent experimental results show that the transcription and splicing machineries are intricately coupled (reviewed by Maniatis and Reed, 2000) In particular, positive feedback exists between the transcription and splicing machineries in which transcription promotes splicing, and splicing in turns promotes transcription As a result, splicing of introns is considered co-transcriptional (Neugebauer, 2002; Tennyson et al., 1995; Wuarin and Schibler, 1994; Bentley, 2002; Cook, 1999; Maniatis and Reed, 2000; Proudfoot et al., 2002; Goldstrohm et al., 2001; Eperon et al., 1988), as it happens simultaneously during transcription of the pre-mRNA, at the point when an exon and its flanking introns are recognized in the nascent transcript (Beyer and Osheim, 1988; Osheim et al., 1985) Co-transcriptional splicing of dystrophin gene was first reported by Tennyson et al (1995) in which the authors observed that “spliced transcript accumulates first at the 5’ end of the gene and at progressively later times as one moves further downstream from the muscle promoter” over a time period consistent with co-transcriptional splicing The authors 127 argued that given the exceptionally large size of the gene and large numbers of exons, co-transcriptional splicing is an effective way to limit the number of possible splice sites and thereby decrease the probabilities of incorrect splicing Co-transcriptional splicing requires exons to be identified co-transcriptionally by splicing factors as well Specifically, by being tethered to both the RNA polymerase II and transcription elongation factors, splicing factors are localized directly adjacent to the nascent pre-mRNA emerging from the polymerase This indicates that co-transcriptional exon recognition occurs at the proximity of the emerging nascent transcript, which seems to be supported by identical observations of both Aartsma-Rus et al (2005) and Wilton et al (2007) They reported that AONs targeting ESE sites in the first half of the exon are generally more efficient in inducing exon skipping than at the other half This suggests that co-transcriptional exon recognition not only occurs as soon as recognition sites are transcribed, it is efficient too; this implies that competition for binding to exon recognition sites is cotranscriptional that starts as soon as they are transcribed Together with the fact that co-transcriptional exon recognition precedes co-transcriptional intron removal (splicing), effective AONs must bind to their target sites during co-transcriptional target exon recognition 128 6.3.1 Consequence of co-transcriptional exon recognition To be efficacious, an AON must bind to an effective target site at the right time An effective target site is a pre-mRNA sequence containing functional ESEs within the exon to be skipped The right time or ‘opportune period’ is before splicing factors bind to the AON target site Thus, two major factors defining AON efficiency are (1) binding to functional ESEs within the target site by the AON and (2) accessibility of the target site to binding during the ‘opportune period’, which in turn depends on the secondary structure of the pre-mRNA The tendency to form complementary base pairings among the nucleotides within the pre-mRNA may cause a target site to be inaccessible, as a nucleotide that is “paired” is not accessible for binding However, there are certain regions in the pre-mRNA with secondary structure motifs devoid of base pairing, such as loops, bulges, joint sequences and free 3’ or 5’ ends (Nowakowski and Timoco, 1999) However, an important consequence of being co-transcriptional is that newly transcribed nucleotides constantly alter the base pairings among the nucleotides of the elongating pre-mRNA, which leads to dynamic and transient secondary structures (Boyle et al., 1980; Kramer and Mills, 1981; Repsilber et al., 1999; Harlepp et al., 2003; Meyer and Miklos, 2004) Therefore, co-transcriptional pre-mRNA folding causes the co-transcriptional binding accessibility of AON target sites to be dynamic and transient The effect of dynamic co-transcriptional pre-mRNA secondary structures on the co-transcriptional binding accessibility of AON target sites has not been studied In fact, no correlation between binding accessibility of AON target sites 129 with efficiency of selective exon skipping is reported (Aartsma-Rus et al., 2005) This perplexing result might be due to the omission in the consideration of the cotranscriptional dynamic and transient binding accessibility of AON target sites 6.4 Summary The use of AON for selective exon skipping as a therapeutic strategy for DMD has progressed to clinical trials To address the many mutations exhibited by DMD patients, an efficient and systematic methodology to determine AON target sites that could induce efficient exon skipping is needed This could not be achieved due to the lack of known major factors affecting the efficiency of exon skipping One such factor is hypothesized to be the dynamics of co-transcriptional binding accessibility of AON target site Because exon recognition is co- transcriptional, an effective AON must bind to its target site to block splicing factors while transcription is ongoing However, the co-transcriptional binding accessibility of the target site is dynamic due to co-transcriptional pre-mRNA folding Given that co-transcriptional splicing of the dystrophin pre-mRNA has been reported experimentally, dynamics of co-transcriptional binding accessibility of AON target site might contribute significant influence on the efficiency of exon skipping, which has not been investigated thus far In the next chapter, it will be shown that dynamics of co-transcriptional binding accessibility of AON target site correlates with efficiency in the induction of selective exon skipping 130 ... 53 55 57 59 61 63 65 67 69 71 73 75 77 79 Figure 6- 1B Percentage length of each exonic region relative to its flanking intronic region To illustrate the fact that the exonic region of the dystrophin... in Figure 6- 1 121 1,000,000 N u m b e r o f b a s e p a irs 100,000 10,000 1,000 100 10 1 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Exon 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75... (Pramono et al., 19 96; Matsuo, 19 96; Matsuo and Takeshima, 2005; Wilton and Fletcher, 2005a, 2005b; Wilton et al., 2007; AartsmaRus et al., 2002, 2004, 2005, 20 06; van Deutekom and van Ommen, 2003;