Journal of Rashid Latif Medical College 1 (2012) 16-32
SHID LATIF RA
Journal of Rashid Latif Medical College
Review Article
j o u r n a l h o m e p a g e : w w w. j r l m c . o r g
Spliceotherapy: Targeting Spliceosome Assembly to Treat Spliceopathies Faiz-ul Hassan Nasim1,2,*, Samina Ejaz1, Asma Yaqoob1 and Muhammad Ashraf2 Department of Chemistry, 2Department of Biochemistry and Biotechnology, Baghdadul Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur – 63000, Pakistan 1
KEY WORDS:
ABSTRACT
Cancer Spliceosome Spliceopathy Spliceotherapy Splicing
Eukaryotic living systems avail post-transcriptional alternative pre-mRNA splicing schemes to generate diverse but closely related tissue-specific and/or cell-stage-specific protein molecules. These events involve manipulation of the splicing process through a variety of combinatorial strategies that involve a number of cis- and trans-acting components. Generally, these strategies target spliceosome assembly on a particular set of donor and acceptor splice sites present in a specific segment of the select pre-mRNA molecule. Normally, the process results in production of the required mRNA and hence the protein variant. However, occasionally the process takes an undesired twist to produce a defective mRNA and hence a corresponding abnormal protein variant. Such unusual splicing schemes have been documented as the cause of various human physiological disorders, recently termed as spliceopathies. The only way to rectify such a disorder is to revert the abnormal splicing pattern back to the normal scheme, an uphill task that can safely be referred to as Spliceotherapy and is currently being negotiated by only a few research laboratories investigating regulation of alternative pre-mRNA splicing schemes. Using published literature as a source, this article will review the alternative pre-mRNA splicing schemes; the splicing aberrations that are known to result in a spliceopathy; and the success and shortcomings of the attempts made so for, spliceotherapy, to cure the corresponding disease. alternative splicing added a further twist in the pre-mRNA splicing scheme suggesting that a single eukaryotic gene could produce multiple diverse albeit closely related protein molecules to fulfill the need of the cell. Soon the scientific community realized that a variety of normal and abnormal alternative splicing schemes exist in nature and while the normal alternative schemes are essential for the survival of a particular cell/tissue, the abnormal scheme could be associated to a particular disease which can technically be referred to as spliceopathy. We now know that the spliceosome is a large ribonucleoprotein complex made up of five Uracil rich small nuclear RNAs-containing particles (the snRNPs) and at least 100 different protein molecules some of which could be assigned to Sm, hn and SR families of proteins. These components referred to as the trans-acting splicing
Split Gene Discovery and Pre-mRNA Splicing: Discovery of split genes by Philip Sharp and colleagues followed by post-transcriptional pre-mRNA splicing in eukaryotic systems provided a new insight into and improved our understanding of the gene expression and regulation processes. The information that the coding sequences could be present in segmented units called exons separated by long stretches of non-coding regions known as intervening sequences or introns was fascinating in itself as it intrinsically pointed towards the presence of a complex machinery, later characterized as spliceosome, capable of identifying exact boundaries besides precisely cleaving these boundaries followed by joining of the exon ends to generate a contiguous unit of coded genetic information that could finally be translated into a functional protein molecule. Later, the discovery of
Correspondence to: *Nasim FH, 1Department of Chemistry, 2Department of Biochemistry and Biotechnology, Baghdadul Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur – 63000, Pakistan.
[email protected]
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surrounding exons to be ligated. Pre-mRNA processing is a co-transcriptional activity and the C-terminal domain of RNA polymerase II is believed to play a critical role in launching spliceosomal components to the cis-acting elements present in the premRNA molecule. Recognition of the splice site sequences may also be aided by a combinatorial contribution of some unique cis-acting elements categorized as splicing enhancers (SEs) or silencers (SSs) and specific transacting factors that bind the particular SEs and SSs and modulate splicing. SEs and SSs are diverse but discrete sequences present within exons or introns, and designated as Exonic Splicing Enhancers/Silencers (ESEs/ESSs) or Intronic Splicing Enhancers/Silencers (ISEs/ISSs), respectively. Many human genetic diseases have been predicted to be linked to mutations in ESEs. In general, the cis-acting elements (splice sites, enhancers or silencer sequences) are recognized as soon as they are transcribed and so one would expect splicing proceed smoothly to end up into an exon-only mRNA product. Such an mRNA, a constitutively spliced message, originates from what is termed as a constitutive splicing event. However, in reality not many pre-mRNAs splice only constitutively. A great majority of the pre-mRNAs follows alternative splicing; a major mechanism that leads to proteome diversity through synthesis of several closely related protein molecules from a single gene. Genetic, developmental or environmental factors including components of spliceosome have been found to be involved in alternative splicing of various gene transcripts. Attempts have been made by various workers to categorize different alternative splicing schemes although none is universally agreed upon. For clarity of discussion and to simplify concepts, we have opted for a generalized system of extension, retention, deletion, truncation etc. Often confusions arise when alternate transcripts originating from different promoters are discussed with the ones that are produced through alternative splicing. In this article we will discuss different aspects of the pre-mRNA splicing process using a primary transcript originating from just one promoter as a model system. All alternative splicing schemes discovered todate can be grouped into the following five broad categories (Figure 2).
factors recognize a variety of short conserved sequences/regions (splice sites, enhancers and silencers) referred to as the cis-acting elements to assemble the spliceosome. The constitutive or the alternative splicing schemes therefore result from combinatorial selection of the cis-acting elements by the trans-acting factors. This understanding has lead to the idea that, in certain cases at least, using a suitable technique referred to as spliceotherapy the abnormal splicing pattern can be reverted back to normal to cure a spliceopathy. In this review article, we have explored rational of the technique and the recent progress in the field.
Spliceosome Assembly, Constitutive and Alternative Splicing: Spliceosome assembly is generally believed to proceed in a step-wise manner and involves five UsnRNAs (U1, U2, U4, U5 and U6) along with a number of proteins referred to as the Splicing Factors (SFs). Processing of the pre-mRNA however starts with the release of U4 snRNP and replacement of U1 snRNP that brings U5 and U6 snRNPs in close proximity to the 5'splice site canonical sequence of AG:GUAAGU. U6 snRNA, believed to be the catalytic ribozyme, then helps in the two transesterification reactions that result in the release of intron in form of a lariat and consequent ligation of the two exons (Figure 1).
Figure 1: Spliceosome assembly and the pre-mRNA splicing proceed in a step-wise manner. Spliceosome assembly involves binding of U1 snRNP to the 5' splice site of the first exon, recognition of the branch point sequence along with the polypyrimidine tract and the AG dinucleotide, collectively termed as the 3' splice site region, by a number of spliceosomal factors including U2 Auxiliary Factor (U2AF), U2 snRNP, and hnRNP I (formerly known as Polypyrimidine Tract Binding Protein or PTB), a tri-snRNP complex comprising U4, U5 and U6snRNPs along with several other proteins then joins to assemble a spliceosome encompassing an intron that is to be excised for removal and partial sequences from the
Figure 2: Alternative pre-mRNA splicing schemes. Splicing events are shown with the help of dotted lines. (a) Constitutive and alternative splicing; (b) Exon 17
truncation/intron extension; (c) Exon extension/intron truncation; (d) Mutually exclusive exons; and (e) Intron retention.
Exon Skipping or Deletion – Cryptic Exon: An exon present somewhere in the middle of a multiexon pre-mRNA behaves as an optional exon. Its inclusion as a unit (cassette) produces a constitutively spliced mRNA while its exclusion results in an alternate transcript and hence an alternate protein molecule. The inclusion or exclusion of the optional exon is accomplished by preferential selection of a particular donor or acceptor splice site from among a pair of proximal (closer) and distal (farther) sites. In a parallel situation, a segment from within an intron may sometimes be included in the final message as a coding unit. Such a segment is designated as a cryptic exon and can be differentiated from a cassette exon on the basis of its frequency of usage with the most frequently used unit being termed as the cassette or constitutive exon. a) Exon Truncation – Intron Extension: An exonic unit in a pre-mRNA may contain multiple donor or acceptor splice sites designated as constitutive or cryptic splice sites on the basis of the frequency of usage with the most frequently used site considered as the normal or constitutive splice site. Sometimes, the competing donor or acceptor splice sites, the cryptic sites, present within the exonic sequences are also categorized as distal or proximal sites based on their position from the corresponding end. Selection of these interior sites results in loss of nucleotides and hence shortening of the coding or lengthening of the non-coding region, a situation that may be referred to as exon truncation or intron extension. b) Exon Extension – Intron Truncation: The cryptic splice sites are present in the intronic sequences, their selection results in gain of nucleotides and hence lengthening of the coding or shortening of the noncoding region. This situation may be referred to as exon extension or intron truncation. c) Mutually Exclusive Exon: Two consecutive or adjacent exons may never appear simultaneously in the final transcript. Inclusion of one member of the pair always results in exclusion of the other. Such exons are referred as mutually exclusive exons. d) Intron Retention: An intronic unit is sometimes included in the final message resulting in conversion of two exons along with their intervening sequence in to a lengthened exonic unit. This situation is referred to as intron retention and usually results in incorporation of new stop codons and hence truncated protein molecules.
Interacting Protein 1 (SIP1) that plays a crucial role in the biogenesis of most of the spliceosomal snRNPs has been implicated in Spinal Muscular Atrophy (SMA), a leading genetic cause of infant mortality, the motor neuron degenerative disease. Similarly, deficiency of SMN protein produces unexpected cell type-specific effects on the selection of snRNAs and mRNAs, causing widespread pre-mRNA splicing defects in many transcripts originating from diverse genes. Preferentially affected are the pre-mRNAs with a large number of introns. Presence of three novel splicing variants of SIP1 in varying concentrations has been reported in normal cells. The fulllength variant SIP1-alpha is expressed at high levels in normal tissues however its expression decreases in the diseased cells. Conversely, SIP1-beta and –gamma are expressed at very low levels in the normal tissues but their expression significantly increases in the tissues of the patients suffering from either SMA or Amytrophic Lateral Sclerosis (ALS). Since SIP1 interacts with SMN protein during snRNP biogenesis, the deficiency of its functional form, SIP1 alpha, severely affects snRNP biogenesis, spliceosome assembly and hence the pre-mRNA splicing process. SMA may originate from deficiency of SMN or SIP1 protein. Deficiency of any of these proteins affects biogenesis of snRNPs. Spinal Muscular Atrophy (SMA), the autosomal recessive neuromuscular disorder caused by a lack of SMN protein is considered to be the most common lethal autosomal disorder among Caucasians children after cystic fibrosis. SMN protein is encoded by SMN1 (Survival Motor Neuron 1) gene and SMA is caused by homozygous deletion of the SMN1 gene. SMN2 is a duplicate copy of SMN1 but it is unable to compensate for the homozygous deletion or absence of SMN1. In fact, SMN2 gene has a CU mutation at position 6 in exon 7 disrupting an ESE and promoting the exclusion of exon 7 in major fractions of SMN2 mRNAs. Severity of the disease is enhanced with the increased extent of abnormal splicing pattern of SMN2 mRNAs. Exon 7 lacking mRNAs are translated to produce truncated and nonfunctional proteins missing 16 amino acids at the Cterminus. Amongst all the tissues studied the brain has the largest amount of alternative splicing activity and is consequently the most affected tissue. Specific splicing abnormalities have been reported in a wide range of neuropsychiatric diseases such as psychotic, affective, bipolar and major depressive disorders, suicide, substance abuse disorders and neuro-developmental disorders. In the brains of dementia sufferers the neuronal cytoskeleton is disturbed due to aberrant tau expression. This protein binds and organizes microtubules to form and maintain neuronal axons. Multiple isoforms of the tau protein generated through alternative splicing of the neuronspecific tau transcript are involved in modulation of its function. Regulation of tau transcript splicing is therefore vital for neuronal health and correct brain function.
Glutamate uptake in the brain is modulated by Excitatory Amino Acid Transporter 2 (EAAT2). This protein is implicated in normal physiology of learning and memory. Its dysregulation is associated with multiple psychiatric and neurological disorders. This molecule has a complex pattern of alternative splicing that produces three coding isoforms and multiple 3'- and 5'-UTRs. These UTRs have been proposed to contain regulatory sequences involved in modulation of EAAT2 expression. Development of psychotic disorders like Schizophrenia (SCZ) and Bipolar Disorder (BPD) are due to alternative splicing of the beta(2)-subunit of gamma-aminobutyric acid (GABA) type A receptor gene (GABRB2) transcript. Non-coding single nucleotide changes in this gene have been associated with SCZ, BPD, mRNA expression and alternative splicing. Depending on intron sequences alternative splicing in Exon 10 region of GABRB2 gene transcript produces multiple isoforms including beta(2S1) and beta(2S2) variants. The beta(2S1) expression decreases while the beta(2S2) expression increases in both SCZ and BPD compared to controls. Thus some noncoding sequences in Exon-10 region play a critical role in the beta(2)-subunit splicing diversity and hence the etiologies of SCZ and BPD. Similarly, Neurofibromatosis type 1 (NF1), one of the most common inherited human disorders, is caused by mutations that lead to protein truncation through alternative splicing. These mutations do not involve the conserved AG/GT sequences of the splice sites but generate frameshift and incorporate nonsense and missense codons. Alternative splicing of Cellular Adhesion Molecules (CAMs) RNAs regulated by trans-synaptic interactions generates countless protein isoforms and ultimately determines neurotransmitter phenotype. These isoforms are required for specialized cell-to-cell connectivity and their absence results in disruption of glutamatergic and gamma-aminobutyric signaling and loss of activitydependent neuronal plasticity. Regulatory polymorphisms switching constitutive and alternative splicing have been suggested as risk factors for numerous neuronal diseases including autism spectrum disorders. Phosphatase and Tensin (PTEN), a tumor suppressor gene located on chromosome 10q23, produces Phosphatase and Tensin homologue protein. Mutations in this gene have been identified in patients with Cowden Syndrome (CS) and Bannayan Zonana syndrome (BZS), two autosomal dominantly inherited diseases, with exon 5 being the most common site of mutation. Agarwal et al., (2005) have investigated the effect of different mutations in PTEN homologue protein. Five distinct splice site mutations led to the skipping of exon 3, 4 or 6 and as a result reduction in dual phosphatase activity of PTEN. Protein phosphatase activity was found to be associated with exon 3 whereas lipid phosphatase activity was found to be associated with exon 4. While 11 point mutations at the splice junctions of the PTEN gene have been reported, 19
three novel splice mutations have been identified in CS and BZS patients. One of these affects a splice-acceptor site and results in out-of-frame skipping of an exon, whereas the other two affect the splice-donor sites and lead to exon extension/intron truncation due to activation of cryptic splice sites. Development of Wilm's tumor (WT) and Acute Myeloid Leukaemia (AML) also involves multiple spliceoforms of an antisense transcript WT1-AS of WT1 gene. Abnormal splicing of the WT1-AS transcript has been suggested in the development of AML malignancy. Misregulation of pre-mRNA splicing associated with the splicing regulatory elements or mutations in the processing factors have been reported to affect expression of tumor suppressors or oncogenes resulting in a variety of carcinomas such as breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, etc. In colorectal cancer, for example, the c-myc transcriptional suppressor, far-upstream element (FUSE)-binding protein (FBP)interacting repressor (FIR), is alternatively spliced. This has been found to be associated with changes in SAP155, a subunit of SF3b splicing factor. Knockdown of this factor leads to the production of a novel splicing variant of FIR which lacks c-myc repression activity. Similar splice variants were also found to be activated in human colorectal cancer. It has therefore been suggested that overexpression of FIR and its splicing variants in colorectal cancer generates its effects via c-myc transcription regulation. AUG containing exon deletion during splicing leads to complete loss of the expected protein. An example of this is the absence of protein 4.1R in red blood cell membrane, a very rare condition in humans. This is caused by a 50 kb genomic DNA deletion which suppresses exon 2 and 4, the two key exons containing functional translation initiation sites in erythroid and non-erythroid cells, respectively. The skeletal muscle Myotonic Dystrophy (DM), the most common form of muscular dystrophy in adults, is another novel RNA splicing-mediated disease. Dysregulation of alternative splicing has been identified as one reason of the pathogenesis of DM type 1 or Steinert's disease. Using RTPCR and protein truncation test (PTT) analysis four splicing related mutations have been identified in patients with Becker or Duchenne muscular dystrophy. One, a 3' splice site mutation (IVS 74-2G), results in a complex pattern of exon skipping involving exons of the C-terminal domain. The other three, consisting nucleotide substitutions in splice donor (IVS26+2TA, IVS65+1GA) or acceptor (IVS8-15AG) sites lead to the use of cryptic splice sites and hence insertions of intronic sequences in the final mRNA. It has been noticed that up to 34% of the point mutations reported in the dystrophin database (http://www.dmd.nl) are splice site mutations although altered splicing has been experimentally confirmed in only 23% of the cases. Different factors are involved in RNA repeat expression in DM patients. Alternative splicing
factor muscle blind-like 1 (MBNL1), for example, binds to the CUG or CCUG repeats in RNA causing splicing misregulation. Several alternative exons including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9 splice aberrantly in human DM1 brain and it has been proposed that the disease symptoms may be alleviated by releasing the CUG or CCUG repeat-bound MBNL1. While mutations in the highly conserved GT/AG splice site dinucleotides are expected to be pathogenic, many intronic mutations do not produce abnormal proteins. According to current estimates, about 5-10% of genomic variants found in familial hypercholesterolemia, F H D / Ta n g i e r d i s e a s e a n d f a m i l i a l hypobetalipoproteinemia are located in the introns of the candidate genes and act as splicing mutations. Defects in Low Density Lipoprotein Receptor (LDLR) lead to Familial hypercholesterolemia (FH), a common single gene disorder that predisposes to coronary artery disease. It has been shown that 14% of LDLR defects are due to splice site mutations. Variations in alternative splice site selection have also been associated with Cystic Fibrosis (CF) severity. Of the 1795 reported mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that cause CF, 221 (12.31%) lead to defective processing of the CFTR pre-mRNA. Alternative splicing has also been observed in the metazoan immune system genes. These diverse and flexible systems have evolved for efficient recognition of “self” and “non-self”. Abnormal alternative splicing leads to malfunctioning of the immune system, inappropriate reaction to self-antigen and hence autoimmune diseases. Netherton Syndrome (NS), a severe skin disease is caused by loss-of-function mutation in Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene. This gene encodes a serine protease inhibitor LEKTI (Lympho Epithelial Kazal Type-related Inhibitor). The loss-of-function mutation disrupts an ESE leading to out-of-frame skipping of exon 11. Similarly mutations in the Dentin Sialophosphoprotein (DSPP) that lead to skipping of exon 3 and activation of a cryptic donor splice site have been recognized as the causative agent of hereditary dentin defects. Retinitis Pigmentosa (RP), an inherited eye disease characterized by progressive degeneration of rod photoreceptor cells, is the example of a disease that has been attributed to mutations in various pre-mRNA splicing factors (PRPFs) including PRPF31, PRPF3 and PRP8. Development of Ataxi-Telangiectasia (AT), also known as Louis-Bar Syndrome, a rare neurodegenerative inherited disease causing severe disability, has been linked to mutations in the ATM gene through exon deletion. Similar linkages of abnormal alternative splicing have been established with many other disorders such as inherited skin disease Dystrophic Epidermolysis Bullosa, Osteopetrosis, Long QT Syndrome type 2, AxenfeldRieger Syndrome (ARS) and a variety of carcinomas. 20
unique role to play, it is extremely important to design drugs that specifically inhibit or block the activity of a particular isoform without affecting the other. Moreover, all isoforms are not equally responsive to various drugs as it is evident by the fact that isoform expressed in the cerebral cortex (COX3) is more responsive to analgesics and antipyretics like phenacetin and its metabolite acetaminophen. Acetaminophen is an NSAID but it does not exhibit profound anti-inflammatory activity in most of the tissues. Inhibitors targeting a particular isoform of COX enzyme are now being preferred to treat pain and inflammation after identification of the isoform involved in the pathogenesis of a disease. Contrary to the approach of inhibiting the activity of altered proteins, pharmacological agonists can be designed to enhance the activity of various protein isoforms. To the best of our knowledge till now this approach has not been applied practically.
Spliceotherapeutic Approaches to cure Spliceopathies: As discussed above and reviewed elsewhere, a wide range of human diseases are caused by alterations in the splicing pattern of different genes. Enhanced knowledge of alternative splicing can help in diagnosis and be employed for developing targeted drugs. Considering the importance of this issue, scientists are in constant efforts to identify therapeutic targets and design appropriate strategies to correct splicing defects. Since disease related defective splicing is mostly the outcome of mutations in splice sites, regulatory sequences or splicing factors, splicing defects can be corrected by targeting either mutated gene sequences (cis-elements) or the factors binding to these sequences (trans-elements). While primary objectives of RNA based therapeutic strategies are, to alter the pattern of target pre-mRNA processing, reprogram genetic defect by repairing defective mRNA and targeting silencing of the defective transcripts, different approaches including the use of small molecules as drugs, RNA based gene therapy or drugs that can inhibit or block the activity of altered proteins have been proposed to rectify defective splicing pattern. In the following section we review a variety of spliceotherapeutic techniques developed to repair defective splicing noticed in many diseases although the job is not yet finished.
ii) Application of Monoclonal antibodies to Target Defective Protein Isoforms: Insertion, exclusion or truncation of exons and retention of introns among many other splicing events change open reading frame (ORF) and transcript producing proteins containing modified domains. Monoclonal antibodies capable of targeting such unique regions of the pathogenic protein isoforms can be used to inhibit/cure a disease. This approach has been tested successfully against tumor specific isoform of CD44 gene, CD44V6. Various CD44 gene products play critical roles in a living system. For example, type 1 transmembrane glycoproteins encoded by CD44 gene mediate cell to cell and cell to matrix interactions. CD44 gene produces 20 different proteins through alternative splicing which undergo a range of post-translational modifications including glycosylation (N- or O-glycosylation) and phosphorylation. Some of these isoforms contain heparin sulphate side chains, can bind with growth factors and regulate growth factor receptor-mediated signaling pathways. One of these isoforms, CD44V6 is the splice variant resulting by the inclusion of exon v6 and it is overexpressed in different types of tumors. Exon v6 specific antibodies have been observed to reduce the concentration of this tumor specific isoform and when used in combination with radiotherapy these antibodies were found to more specifically kill the target cells and decrease side effects of radiotherapy.
A) Targeting Alternatively Spliced Protein Isoforms: Defective protein isoforms can be blocked using small molecules, drugs or antibodies to correct various disease related splicing defects. i)
Use of Drugs to Inhibit or Block Protein Isoforms: Splice variants lead to the production of different protein isoforms having unique properties. Thus isoformspecific small molecules that specifically inhibit/block activity of the unwanted disease associated protein can be designed and explored as drugs to treat splicing defects. An example of this approach is the use of non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit activity of different isoforms of cyclo-oxygenase (COX) Enzyme. COX enzymes catalyze a critical step of metabolic pathway related to prostaglandin synthesis and are encoded by two different genes (PTGS1/COX1 and PTGS2/COX2). Both, COX1 and COX2 are known to undergo alternative splicing. While COX2 is induced by various physiological conditions like inflammation, fever or injury, constitutively spliced transcript of COX1 results in the formation of an isoform which is required for the synthesis of physiologically relevant prostanoids that regulate stomach mucosa and platelet aggregation. Alternative splicing of COX1 however produces COX3 isoform that plays an important role in the control of fever and this is the molecule inhibited by fever specific NSAIDs. Since different isoforms of COX enzymes have
B) Targeting Specific mRNA to Correct Splicing Defect: A variety of strategies have been tested by targeting specific mRNAs to correct disease specific errors in splicing pattern. The target mRNA is either degraded, modified by the removal or addition of exons, and repaired/reprogrammed by adding foreign mRNAs 21
depending upon the need of time.
which target splicing regulators and alteration of misspliced mRNA sequences through trans-splicing. Applications of all suggested therapies along with the merits and demerits of each are summarized below.
1. Elimination of Undesired mRNA: Messenger RNA degrading strategies resulting in elimination of the undesired mRNA include RNA interference (RNAi) and use of antisense oligonucleotides. Both these methods are based on RNase-H mechanism which induces degradation of the targeted mRNA.
a) Small molecules / drugs targeting splicing factors to modulate gene expression: Some spliceosome components have synergistic or antagonistic effects that vary in a concentration dependent fashion. Serine Arginine rich (SR) splicing factors and heterogenous nuclear ribonucleoproteins (hnRNPs), for example, are known to affect selection of competing proximal/distal splice sites and hence the alternative splicing schemes. Altered levels of SR proteins influence transcription of many proteins. Since small molecules or drugs can directly influence the process of gene expression and regulate the concentration of certain proteins, they can modulate splicing of a particular pre-mRNA. To correct splicing defects scientists have either targeted splicing factors directly or the enzymes modifying and activating splicing factors. Differential binding of SR proteins and hnRNPs with specific exonic/intronic sequences affects interactions of the basal splicing machinery and hence modulates choice of splice sites. Furthermore, the phosphorylation status of SR proteins greatly influences their RNA binding specificity, ability to interact with other proteins and intracellular distribution. Clk1/Sty kinase activity that phosphorylates SR proteins has therefore been among the targets to modulate splicing and a novel inhibitor of Clk1/Sty has been shown to affect the ASF/SF2 dependent splicing. Also in the list are the compounds that have the potential to bind with SR proteins and interfere with spliceosome assembly. A case example is that of HIV-1 pre-mRNA splicing which is modulated by various members of the SR proteins family. Its alternative splicing generates 40 different mature mRNAs through different combinations of 5' and 3' splice sites that depend upon interactions between HIV-1-pre-mRNA and various trans-acting elements (SR proteins and hnRNPs). Indole derivative (IDC16) has been shown to interfere with ESE activity of the SF2/ASF SR protein thereby suppressing the production of viral particles by decreasing synthesis of key viral proteins, transcription of full length pre-mRNA and assembly of infectious particles. IDC16 blocks the production of HIV viral particles in peripheral blood mononuclear cells (PBMCs) or macrophages infected with different laboratory strains. It is important to note that IDC16 also reduces the production of HIV viral particles in clinical isolates obtained from patients exhibiting resistance to anti-HIV multi-therapies. Small molecules have also been successfully used to promote the inclusion of exon 7 in SMN2 transcripts and treatment of SMA. Interferons (IFNs) interact with IFNs response elements located in the promoter of SMN genes and stimulate their transcription. Similarly valproic acid treatment stimulates transcription in the fibroblast cell
a) RNA Interference (RNAi) Induced Depletion: RNAi is a powerful tool for the post-transcriptional regulation of gene expression. Small interfering 21-23 nucleotide long RNAs (SiRNAs) having sequences complementary to the target mRNA specifically bind with the target mRNA and the SiRNA-target mRNA duplex thus formed is degraded by dicer resulting in the RNAi mediated silencing of the gene. RNAi has been successfully used to target diseases specific splice variants. A common example is the down-regulation of Bcl-xL mRNA that results in the down-regulation of antiapoptotic Bcl-xL protein. Hutchinson-Gilford Progeria Syndrome (HGPS), a rare genetic disorder known as the disease of pre-mature aging, is characterized as defective splicing disease caused by a heterozygous silent mutation in exon 11 of LMNA (lamin A) gene. Mutation in exon 11 enhances the use of an internal or upstream 5' splice site instead of normal 5' splice site of exon 11 and resulting mRNA leads to the production of truncated protein known as progerin whose accumulation results in premature aging. SR-proteins SRSF1 (serine-arginine rich splicing factor 1) and SRSF6 play an important role in 5' splice site selection of exon 11 and it has been observed that RNAi induced depletion of SRSF1 significantly lowers progerin in HGPS-like mouse embryonic fibroblasts (MEFs) reducing symptoms of HGPS. However, depletion of SRSF6 further promotes the development of HGPS phenotype. b) Antisense oligonucleotides: Antisense oligonucleotides were first used in 1970 by Zamecnik and Stephenson to inhibit Rous sarcoma virus in tissue cultures. Antisense oligonucleotides can bind with the complementary sequences present in the target mRNA, induce RNase H dependent degradation of the target mRNA and efficiently reduce the expression of encoded protein. RNase H dependent oligonucleotides are designed by targeting any region of mRNA but there are issues of effective design and efficient target delivery associated with these oligonucleotides. 2. Reprogramming Splicing: Erroneously spliced mRNAs have also been targeted indirectly through strategies that can reprogram splicing of the corresponding pre-mRNA. This is considered to be an effective approach to correct splicing based diseases. Suggested strategies employ use of small molecules/drugs 22
(obtained from the SMA patients) cultures and increases the ratio of exon 7+ as compared to exon 7- SMN2 transcripts. Sodium butyrate treatment of SMA model mouse also resulted in increased overall transcription. Aclarubicin which is the catalytic inhibitor of topoisomerase II raises the level of SMN protein by increasing the inclusion of SMN2 exon 7 . It is known that SMN1 gene is essential for the assembly of U1 SnRNP complexes in the cytoplasm and loss of snRNA synthesis has been identified as the characteristic feature of SMA. It has been observed that valproic acid raises the level of SR proteins. Hence effects of valproic acid treatments on SMN genes transcription are suggested to be mediated through increased SR proteins concentration. Butyrate and valproic acid inhibit histone deacetylases (HDAC) and increase transcription of many genes. Details are not known about the mechanism of action of aclarubicin except that the treatment of aclarubicin leads to sub-cellular redistribution of SR proteins. Gemcitabine has been reported to increase intracellular levels of ceramide which in turn regulates alternative splicing of caspase 9 and Bcl-x. Steroid hormones have also been found to significantly regulate the splicing pattern of steroid hormone dependent genes transcripts. Thus steroid hormone antagonists are being considered as pharmacological agents. However, as multiple genes are known to be steroid hormone dependent, these antagonists need to be used with caution since they may affect multiple transcripts. Retinitis Pigmentosa, a blinding disease, originates from aberrant splicing of retinitis pigmentosa GTPase regulator (RPGR) and Bardet Biedl syndrome 1 (BBS1) genes. Both these genes have mutated 3' splice sites. Engineered / modified U1 SnRNA has been proven to be helpful for the efficient recognition of mutated 3' splice sites and correction of mutations induced splicing defect in BBS patient derived fibroblasts. Defective U1 SnRNP assembly globally affects the binding of trans-elements with pre-mRNA molecules. Modified or mutated exon specific U1 SnRNAs (ExSpeU1) have been shown to exhibit significant potential to repair splicing defects in haemophilia B, cystic fibrosis and SMA. Splicing defects were identified to be the skipping of F9 exon 5 (haemophilia B), CFTR exon 12 (cystic fibrosis) and SMN2 exon 7 (SMA) caused by mutations in the consensus sequence donor (5') splice site, polyprymidine tract of acceptor site and regulatory elements present in exons. Butyrate has been used in the treatment of sickle cell disease. Similarly valproic acid is categorized as antiepileptic agent. It is very unfortunate that both these reagents modulate global gene expression rather than correcting the mis-splicing of specific genes.
b) Modifying pre-mRNA splicing using anti-sense oligonucleotide-based approach: Modified DNA and RNA oligonucleotides have been used to promote the desired splicing pattern and synthesize required transcripts and protein isoforms109. Such oligonucleotides are termed as splice switching oligonucleotides (SSOs) and can be used for both upregulation and down-regulation of desired protein isoform depending upon what is required at a particular time? Using these oligos aberrant splice sites can be blocked to restore normal splicing whereas alternate splice sites can be targeted to modulate splicing producing detrimental isoforms to the splicing scheme synthesizing beneficial isoforms. Gene specificity is attained by targeting specific cis-elements. SSOs bind with specific cis-elements to block its activity. As a result a specific splice event is either activated or inhibited. In few cases, scientists have used chemically modified (2' O-methyl or a morpholino backbone) SSOs which prevent the degradation of targeted pre-mRNAs. Oligonucleotides provided with morpholino backbone have been used to successfully repair splicing defect in erythroid cells of B-thalassemia/HbE disorder patients and increase the concentration of correctly spliced â-E-globin mRNA in a dose dependent and sequence specific manner. i)
SSOs mediated exon skipping: SSOs may specifically be targeted to bind with the sites essential for the inclusion of an exon in the final mRNA and thus hide this exon from splicing machinery. Thus exon skipping can be mediated with the help of SSOs. This approach has been used to induce a new splice variant of tumor necrosis factor receptor 2 gene (TNFR2). Mild over-expression of a novel splice variant of TNFR2 (lacking exon 7), which is translated to produce soluble isoform of the receptor, has been observed in few inflammatory conditions like collagen induced arthritis (CIA) or TNF-á induced hepatitis. The soluble protein can sequester some of the TNF-á which is responsible for many symptoms of the inflammatory conditions. Efficient SSOs have been designed that induce skipping of exon 7 in TNFR2 pre-mRNAs to increase soluble receptor and subsequently block the TNF-á signal. SSOs have been found to be more effective to reduce the effects of TNF-á as compared to other drugs that are commonly being used to treat inflammatory disorders. SSOs-mediated exon skipping is also one of the most promising therapeutic approaches to correct splicing defect causing Duchenne Muscular Dystrophy (DMD), the outcome of defective splicing of DMD gene producing non-functional dystrophin protein. Two hot spot regions (major: exon 45-53; minor: exon 2-20) have been identified in DMD genes where most of the deletions and duplications occur. Deletions of the exon 48-50 region disrupt open reading frame (ORF) with the generation of a premature termination codon which results in the synthesis 23
of truncated non-functional dystrophin protein. SSOs able to hybridize with exon 51 hide it from splicing machinery and so exon 51 is spliced along with its flanking intron, ORF is restored and shorter but functional dystrophin protein is synthesized. A successful trial of SSOs to correct DMD in humans has been completed. Another independent study has shown that SSOs can be used to treat mdx mice models of DMD. Exon 23 of dystrophin gene contains a non-sense mutation and therefore exon 23 containing transcript is translated to produce nonfunctional dystrophin. Whereas if exon 23 is excluded a short and partially functional dystrophin is synthesized. 2'O-methyl antisense SSOs targeting splice sites of exon 23 induce skipping of this exon. Normal level of dystrophin is regained in the muscle fibers and function of muscles is improved. Myotonia is characterized by reduced conductance of chloride ions, stiffness, increased excitability and delayed relaxation of skeletal muscles in DM. Morpholino antisense oligonucleotides targeting 3' splice site of muscle chloride channel gene 1 (ClC-1) exon 7 have been shown to promote skipping of this exon in ClC-1 mRNA to restore ORF and increase synthesis of normal ClC-1 protein. This protein localized on surface of membranes helps to eliminate symptoms of myotonia in DM. Use of SSOs has also been suggested as a promising tool for the correction of splicing defect in human tau premRNA splicing. Inclusion of exon 10 in human tau mRNA molecule has been associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). SSOs targeting splice junctions of exon 10 have been shown to suppress inclusion of exon 10 in neuronal pheochromocytoma cells and therefore useful for (FTDP-17) therapy.
introducing a consensus ESE have been shown to help achieve normal splicing in cell lines as well as patient derived cells. c) Using bifunctional oligonucleotides to modify premRNA splicing: Oligonucleotides that contain 2 domains, antisense targeting domain, and effector domain which either induce the exclusion or inclusion of an exon are referred to as bifunctional oligonucleotides. Different modifications of this strategy have been used to modulate splicing patterns. i) Exon Specific Splicing Enhancement by Small Chimeric Effectors (ESSENCE): This approach uses hybrid protein nucleic acid (PNA)peptide oligomer to promote inclusion of weak exons that are naturally defective or mutated in a disease. In these oligonucleotides effector domain is the peptide containing 5, 10, or 15 arginine-serine (RS) repeats. Targeted PNA(RS)n oligomers promote the inclusion of exon 18 in BRCA1 mRNA and exon 7 in SMN2 mRNAs. Exon 18 of BRCA1 and exon 7 of SMN2 gene are weak exons containing weak ESE. However, the mechanism of action of this approach is not yet clear and the degree of exon specificity is not determined. PNA-(RS)n have been tested in tissue extracts and there is need to study the efficacy and specificity in living cells and model organisms. There is possibility of non-specific interaction of (RS)n peptides with cellular proteins or ESE/ESS present in non-target pre-mRNAs. ii) Targeted Oligonucleotide Enhancement of Splicing (TOES): TOES employs oligonucleotides containing 2'Omethyl modified binding domain. However, effector domain contains binding site for known splicing factors and hence can indirectly recruit splicing factors to modulate the splicing of target pre-mRNAs. For example, if the effector domain contains sequence 5'-GGAGGA-3' repeated three times oligonucleotide is predicted to recruit ASF/SF2. Cell free in vitro splicing reactions have shown that TOES approach can be successfully used to promote the inclusion of exon 7 in SMN2 mRNAs. Moreover, when ESE containing bifunctional oligonucleotides were transfected in the fibroblasts of SMA patients endogenous concentration of SMN2 exon 7+ transcripts was increased to the normal level with an increase of ~60 to ~80 %.
ii) Using SSOs to block cryptic splice site: Sometimes mutations generate cryptic splice sites that compete with the constitutive sites and therefore generate aberrant splice variants. In case of â-globin gene a cryptic 3' splice site is created as a result of mutation in intron 2 and an additional exon is included in the â-globin mRNAs. SSOs specifically designed to bind and block cryptic acceptor site sequences have successfully been used to guide spliceosome for right splicing. Using this strategy normal HbA is produced in human erythroid progenitor cells obtained from thalassemia patients and sustained correction of â-thalassemia phenotype is achieved after lentiviral delivery of a vector expressing antisense RNA.
iii) Targeted Oligonucleotide Silencing of Splicing (TOSS): Bifunctional oligonucleotides having hnRNPA1 binding ESS as the effector site inhibit the use of alternative 5' ss during BCL2L1 mRNA synthesis and are used to balance the ratio of Bcl-xL/Bcl-xS transcripts in cancer cells. Bcl-xL is the anti-apoptotic isoform while
iii) SSOs mediated exon inclusion: Homozygous deletion of SMN1 gene and the presence of mutated SMN2 gene results in SMA disease. SMN2 gene is unable to compensate for the absence of SMN1 gene due to a mutated ESE present in exon 7. Disruption of ESE is responsible for the skipping of exon 7 in mature transcripts. SSOs masking the mutated ESE and 24
spliceosome which mediates trans-splicing is not known. In addition, it has been observed that in most of the cases all mutations in an affected population cannot be corrected using PTM. SMaRT also depends upon distribution of genes into cells and tissues hence the complex issues of delivery are also associated with SMaRT. SMaRT has mostly been mediated through viral vectors. However, non-viral Sleeping Beauty (SB) transposon system has also been utilized successfully for the stable delivery of specifically designed trans-splicing molecules to correct the splicing defect associated with severe combined immune deficiency (SCID), an immunological disorder that is the consequence of a point mutation in the gene encoding DNA protein kinase catalytic subunit (DNA-PKcs). It is characterized by hypersensitivity to radiation-induced injury, aberrant DNA repair and defective T/B cell production. In another study recombinant adeno-associated virus (rAAV) vectors have been effectively used to restore the function of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) and conductance of chloride using SMaRT technology. CFTR-PTM binds with intron 9 of CFTR-pre-mRNA and endogenous spliceosome mediates the trans-splicing of CFTR exons 10-24 into endogenous CFTR-pre-mRNA in polarized human cystic fibrosis airway epithelial cells.
Bcl-xS is the pro-apoptotic isoform. Lower ratio of BclxL/Bcl-xS promotes apoptosis. However, there is need to evaluate the efficacy of oligonucleotides based strategies in living organisms or animal models and to determine offtarget effects. Similarly issues of specificity and delivery of oligonucleotides in humans have not yet been addressed. d) Correction of splicing defect by altering sequence of mRNA using strategy of trans-splicing: Trans-splicing is a naturally occurring phenomena but it is rarely observed in mammals. It involves splicing between two separately transcribed pre-mRNAs resulting in the formation of one composite mRNA. This approach targets pre-mRNA or nascent transcript to switch splicing from defective to desired pattern for the correction of splicing defects. There are three ways to apply trans splicing to reprogram splicing of defective genes. i) Spliceosome Mediated RNA Trans-splicing (SMaRT): In this method an engineered pre-mRNA Trans Splicing Molecule (PTM) targets and binds with primary transcript through base pairing interactions having antisense effect. PTM contains 5' binding sequence that can specifically bind with the target pre-mRNA, a splicing region containing motifs essential to promote transsplicing reaction and a region having modified exons that replace the defective exons of the targeted pre-mRNA. Binding of PTM with targeted pre-mRNA is followed by the splicing of an exon present in the targeted molecule with an exon present in the PTM. Trans-splicing is carried out by endogenous spliceosome machinery present in the nucleus. The resulting trans-spliced mRNA contains reprogrammed genetic information. It is transported to cytoplasm where it undergoes translation to produce desired protein isoforms. Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe autosomal recessive skin blistering disease characterized by functional defects in type VII collagen caused by pre-mature termination codons present on both alleles of collagen type 7 alpha 1 (COL7A1) gene. SMaRT was used to restore the expression of functional type VII collagen protein in primary keratinocytes derived from an RDEB patient. This approach has also been successfully employed to correct splicing defects in animal models of various diseases including hemophilia A (factor VIII deficiency), X-linked immunodeficiency and cystic fibrosis (CF). SMaRT is highly advantageous approach with many benefits like a gene is repaired instead of introducing new gene so endogenous components are employed for regulation and PTM constructs can be easily accommodated in the available vector systems. Moreover all problems arising from non-specific targeting are not observed using SMaRT. SMaRT has certain limitations as it may yield undesired products, and specificity of the
ii) Ribozyme mediated trans-splicing: Trans-splicing approach helps to produce chimeric transcripts by the splicing of foreign cDNA and targeted mRNA. This strategy involves reprogramming of target mRNA present in the cytoplasm by trans-splicing mediated by group 1 ribozymes. Ribozyme containing trans-splicing molecule is comprised of a 5' guide sequence which is complementary to the target sequence and helps to base pair with the target molecule, group 1 ribozyme domain, and a 3' region having exon that is required to be altered in the target molecule. Once the trans-splicing molecule is bound with target mRNA, ribozyme catalyzes trans-splicing between 5' exon of target molecule and 3' exon of trans-splicing molecule. Expanded repeats present in the 3' untranslated region of Dystrophia Myotonica-Protein Kinase (DMPK) gene are associated with Myotonic Dystrophy (DM) disease. Length of CUG repeats at 3' end of DMPK transcripts has been reduced by employing specifically designed transsplicing ribozymes. This approach has been tested in cell cultures but needs to be evaluated in the animal models. iii) tRNA splicing endonuclease mediated transsplicing: tRNA splicing endonuclease has been isolated from archeon Methanococcus jannaschii and it is capable of carrying out cis- and trans-splicing. Expression of endonuclease and targeting mRNA is essential for reprogramming of the defective transcripts. This approach has successfully been used to correct exogenous luciferase 25
transcripts and endogenous carnitine acetyl transferase (Crat). Targeting mRNA is not only dependent upon base pairing but it is also mediated through a bulge (3 nucleotides)-helix (4 nucleotides)-bulge (3 nucleotides). Target and targeting RNA in bulge are precisely cleaved by archeal endonuclease and trans-splicing reactions are mediated by the endogenous enzymes. As a result two new transcripts are produced, one having 5' end of targeting mRNA ligated to 3' end (mutated region) of target mRNA whereas the second molecule is the reprogrammed mRNA containing 5' end of the target mRNA ligated to 3' end (corrected region) of targeting mRNA. Archeal endonuclease mediated trans-splicing is suggested to be an approach of higher specificity than spliceosome or group 1 ribozyme mediated approach due to its dependency of bulge-helix-bulge structure. However, activity of archeal endonuclease might be compromised in mammalian cells and systems. This approach has not been widely used to correct splicing defects so many aspects of the strategy are not fully explored. e) Using chemical compounds to correct pre-mRNA splicing defects: Conversion of severe DMD into a milder phenotype by controlling dystrophin mRNA splicing has successfully been achieved using small chemicals that enhance exon skipping in a dystrophinopathy patient suffering from a point mutation in exon 31 of the dystrophin gene. This mutation generates a stop codon, promotes exon skipping and leads to production of a small amount of internally deleted but functional dystrophin protein. A small chemical molecule TG003 has been found to promote skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner increasing production of the dystrophin molecule in the patient's cells. The presence of r(CNG)exp triplet repeats dependent pre-mRNA mis-splicing is associated with many neurological and neuromuscular disorders including myotonic dystrophy type 1 (DM1), spinocerebellar ataxia type 3 (SCA3), fragile x-associated tremor ataxia syndrome (FXTAS) and Huntington's disease (HD). All r(CNG)exp triplet repeats form similar hairpin structures having 1x1 nucleotide RNA internal loops that vary among different genes (U/U, A/A, G/G internal loops). PremRNA splicing pattern is altered due to the sequestration of muscle blind-like 1 (MBNL1) splicing factor and formation of r(CNG)exp –MBNL1 complex. A group of workers have screened a range of synthetic chemical compounds for their specificity to bind with RNAs containing 5'CAG/3'GAC motifs and correction of the exp splicing defects arising due to r(CNG) –MBNL1 complex formation. Among the series of tested compounds 4-guanidinophenyl 4-gunanidinobenzoate was found to be the most efficient for improving splicing defects originated through sequestration of MBNL1 by r(CNG)exp in patient derived cell lines and was therefore
suggested to be a better therapeutic tool for the treatment of exp r(CNG) dependent neurological diseases. Familial Dysautonomia (FD), a severe neurodegenerative disorder is characterized by low levels of inhibitor of kappa B-kinase complex associated protein (IKAP). IKAP is encoded by inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complexassociated proteins (IKBKAP) gene. An intronic mutation IVS20+6TC in IKBKAP gene promotes tissue specific skipping of exon 20 which ultimately reduces the level of IKAP. This pre-mRNA splicing defect was corrected in FD mouse model after administration of plant cytokinin kinetin as nutritional supplement.
Conclusion: Efforts are in progress to understand mechanistic details of the alterations in pathogenic splicing defects and develop strategies to modulate splicing patterns for correction of the corresponding disease. However, todate no approach has been completely validated and optimized for clinical use except first-in-man trial that has been successfully completed to check the validity of SSOs to treat DMD patients. While the SSOs based therapies seem to be the most effective, they have certain demerits including low efficacy in specific tissues and poor cellular uptake. In addition, RNA/DNA antisense oligonucleotides are rapidly cleared from circulation and therefore need to be repeatedly administered in order to achieve therapeutic efficacy. SnRNAs mediated shuttling of antisense oligonucleotides has been devised to overcome the limitations associated with subcellular localization of premRNA and for permanent correction of defective splicing. But still the issues of specificity and delivery of oligonucleotides are there to be addressed. Similarly, strategies targeting components of common splicing machinery including splicing factors like U1 SnRNP and regulators like SR-proteins and hnRNP-A1 may influence gene expression globally leading to generate off-target effects. There is need to develop efficient and better strategies to correct disease related splicing defects. Except few, most of the reported studies are based on either in vitro experiments conducted in the soluble extracts or in vivo experiments performed in cell lines cultures. It is therefore necessary to evaluate the specificity and efficiency of already reported therapeutic tools in animal models and ultimately clinical trials should be carried out involving humans.
Acknowledgements: The authors appreciate contributions of Dr. Jameel ur Rahman and the anonymous reviewers in improving the language and the content of the manuscript, respectively.
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