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The pathogenicity of splicing defects: mechanistic insights into pre‐ mRNA processing inform novel therapeutic approaches

2015; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês

10.15252/embr.201541116

1469-3178

Élisabeth Daguenet, Gwendal Dujardin, Juan Valcárcel,

Neurogenetic and Muscular Disorders Research

Review13 November 2015free access The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches Elisabeth Daguenet Elisabeth Daguenet Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Search for more papers by this author Gwendal Dujardin Gwendal Dujardin Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Search for more papers by this author Juan Valcárcel Corresponding Author Juan Valcárcel Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Elisabeth Daguenet Elisabeth Daguenet Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Search for more papers by this author Gwendal Dujardin Gwendal Dujardin Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Search for more papers by this author Juan Valcárcel Corresponding Author Juan Valcárcel Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu-Fabra, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Author Information Elisabeth Daguenet1,2,‡, Gwendal Dujardin1,2,‡ and Juan Valcárcel 1,2,3 1Centre de Regulació Genòmica (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain 2Universitat Pompeu-Fabra, Barcelona, Spain 3Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain ‡These authors contributed equally to this study *Corresponding author. Tel: +34 93 316 01 56; E-mail: [email protected] EMBO Reports (2015)16:1640-1655https://doi.org/10.15252/embr.201541116 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Removal of introns from pre-mRNA precursors (pre-mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. Mutation or natural variation in pre-mRNA sequences, as well as in spliceosomal components and regulatory factors, has been implicated in the etiology and progression of numerous pathologies. These range from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer. Understanding the molecular mechanisms associated with splicing-related pathologies can provide key insights into the normal function and physiological context of the complex splicing machinery and establish sound basis for novel therapeutic approaches. Glossary AD Alzheimer's disease ALS Amyotrophic lateral sclerosis AML Acute myeloid leukemia APC2 Adenomatosis polyposis coli 2 APP Amyloid precursor protein AS Alternative splicing ASO Antisense oligonucleotide BPS Branch point sequence BRAF V-Raf murine sarcoma viral oncogene homolog B BRR2 Bad response to refrigeration 2 CFTR Cystic fibrosis transmembrane conductance regulator CLL Chronic lymphocytic leukemia CLCN1 Chloride channel, voltage-sensitive 1 CMML Chronic myelomonocytic leukemia CNBP CCHC-type zinc finger, nucleic acid-binding protein CUG-BP1 CUG-binding protein 1 DM Myotonic dystrophy DMD Duchenne muscular dystrophy DMPK Dystrophia myotonica-protein kinase EFTUD2 Elongation factor Tu GTP binding domain containing 2 ESE Exonic splicing enhancer ESS Exonic splicing silencer ExSpeU1 Exon specific U1 snRNA FD Familial dysautonomia FTDP-17 Frontotemporal dementia and parkinsonism linked to chromosome 17 FTD Frontotemporal dementia FTLD Frontotemporal lobar degeneration FUS Fused in sarcoma GOF Gain of function HD Huntington's disease hMpn1/Usb1 Mutated in poikiloderma with neutropenia protein 1/U6 snRNA biogenesis 1 hnRNP Heterogeneous nuclear ribonucleoprotein Htt Hungtingtin HuR Hu antigen R IKBKAP Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein ISE Intronic splicing enhancer ISS Intronic splicing silencer LOF Loss of function MAPT Microtubule associated protein Tau MBNL Muscleblind MDS Myelodysplastic syndrome MFDM Mandibulofacial dysostosis with microcephaly MOPD1 Microcephalic osteodysplastic primordial dwarfism type 1 Myc V-myc myelocytomatosis viral oncogene &!#6;homolog NTC NineTeen Complex NOVA Neuro-oncological ventral antigen ORF Open reading frame PAP-1 Pim-1-associated protein PKC Protein kinase C PN Poikiloderma with neutropenia PRP40 Pre-mRNA processing factor 40 PRPF Pre-mRNA processing factor PTB Polypyrimidine tract-binding protein PTM Pre-trans-splicing molecule RARS Refractory anemia with ring sideroblasts RAS Rat sarcoma gene RBFOX RNA-binding protein Fox RBM5 RNA-binding motif protein 5 RNA-Seq RNA sequencing Ron Recepteur d'origine nantais oncogene RP Retinitis pigmentosa S6K1 Ribosomal protein S6 kinase I Sam68 Src-associated in mitosis 68 kDa protein SAV Splice-altering variants SF1/BBP Splicing factor 1/branch point binding protein SLE Systemic lupus erythematosus Sm Protein components of many snRNPs, named in honor of S. Smith, a SLE patient SMA Spinal muscular atrophy SMART Spliceosome-mediated RNA trans-splicing SMN1 Survival motor neuron 1 snRNP Small nuclear ribonucleoprotein SNRNP200 Small nuclear ribonucleoprotein 200 kDa (U5) SNW1 SNW domain containing 1 SR proteins Serine/arginine proteins SS Splice site SSO Splice-switching oligonucleotide TARP Talipes equinovarus, Atrial septal defects, Robin sequence, and Persistent left superior vena cava TDP-43 Transactive responsive DNA-binding protein 43 kDa TIA-1/TIAR T-cell-restricted intracellular antigen-1/TIA-1-related protein U2AF U2 auxiliary factor UBL5 Ubiquitin-like protein 5 U snRNP Uridine-rich small nuclear ribonucleoprotein ZNF9 Zinc finger protein 9 ZRSR2 Zinc finger (CCCH type), RNA-binding motif, and serine/arginine-rich 2 Introduction The central dogma of Molecular Biology emerged originally as a collinear view of gene expression, in which the information flows from DNA to protein through messenger RNA (mRNA) molecules. The last decades of research have considerably expanded this paradigm by showing the multiplicity of transcripts that can be generated from a single DNA locus through the use of alternative promoters, termination sites, and through alternative splicing of introns, intervening sequences present in primary transcripts that need to be removed to generate translatable mRNAs. Furthermore, intertwined links between transcriptional and posttranscriptional steps in the gene expression pathway, both in the nucleus and in the cytoplasm, not only facilitate coupling between these processes but also expand their regulatory possibilities, particularly in higher eukaryotes 1. Pre-mRNA splicing requires precise recognition of cis-acting sequences on the pre-mRNA by spliceosomal components and additional RNA-binding factors, and involves a vast network of RNA–RNA, RNA–protein, and protein–protein interactions. The realization that at least 95% of human genes produce multiple spliced RNA species via alternative exon usage has revealed the prevalence of this additional layer of gene expression regulation 2. Indeed, alternative splicing (AS) enables individual genes to increase their coding capability and to generate a set of structurally and functionally distinct protein isoforms. The main types of AS are “cassette” exon skipping, alternative 5′ and 3′ splice site selection, alternatively retained introns, and mutually exclusive exons. Interestingly, the frequency of AS varies with species complexity and cell type, during development or upon cellular differentiation, thereby participating in the fine tuning of a gene signature both temporally and spatially 34. Mis-regulation of splicing has been long known to be related to an increasing number of human pathologies, including genetic diseases, neurodegenerative disorders, and cancer. Alterations in pre-mRNA splicing can either act as drivers of disease etiology or act as modifiers that sensitize individuals to disease susceptibility and severity. Recent excellent reviews have covered multiple aspects of this topic 567891011. In this review, we provide a general overview of the function of the spliceosome and the combinatorial rules governing the splicing code. Our focus will be on splicing aberrations in various pathological contexts and how understanding the underlying mechanistic principles can set the stage for the development of novel therapeutic approaches and at the same time shed light on the function and physiology of splicing itself. Basics of the pre-mRNA-splicing process Successful completion of the splicing reaction and deployment of its physiological function require both fidelity and flexibility. First, the discrimination between correct and incorrect splice sites is achieved through systematic, multistage proofreading of the sequences by different factors. Second, splicing commitment is subject to an elaborated and dynamic crosstalk between splicing regulatory factors in order to enforce or to repress splice site selection (for recent reviews: 1213141516). Exon definition & the spliceosome assembly pathway Intron removal is orchestrated by the multi-megadalton macromolecular ribonucleoprotein complex known as the spliceosome, which is composed of five small nuclear ribonucleoproteins (U1, U2, U4/U6, U5 snRNP) and more than 200 snRNP- and non-snRNP-associated proteins 14. The definition of an intron relies on four consensus elements: the exon/intron junctions at the 5′ and 3′ end of the intron—the 5′ and 3′ splice sites (SS)—, the branch point sequence (BPS) located upstream of the 3′ SS, and the polypyrimidine tract located between the BPS and the 3′ SS (Fig 1A). The BPS adenosine plays a crucial role in splicing catalysis, by forming a 2′–5′ phosphodiester bond with the 5′ end of the intron after the first step of the reaction. The 5′ SS and the region surrounding the BPS are recognized through base-pairing interactions with U1 and U2 snRNAs, respectively. Additional regulatory sequences within introns and exons contribute to splice site recognition by the core splicing machinery (Fig 1B and see below). In vertebrates, given the longer length of introns compared with exons, splice sites flanking an internal exon communicate with each other to help in initial exon definition 17, and subsequently engage in interactions across the intron to allow intron removal and exon inclusion (Fig 1C). Modulation of splice site pairing during exon and intron definition can be the target of regulators 181920 (Fig 1C). Figure 1. Mechanisms of splice site recognition and exon definition(A) Splicing complex assembly is initiated by consensus sequence elements located at the exon (blue)/intron (brown) boundaries. Recognition of the 5′ SS by U1 snRNP involves base-pairing interactions between the 5′ end of U1 snRNA. Recognition of the 3′ SS region involves binding of the U2AF65/35 heterodimer to the polypyrimidine tract (Poly-Y tract) and conserved 3′ SS, which facilitates recruitment of U2 snRNP to the branch site, involving base-pairing interactions between U2 snRNA and nucleotides flanking the branch point adenosine. (B) Exon definition modulated by exonic and intronic sequence elements, which can promote (ESE & ISE, in orange) or suppress (ESS & ISS, in dark red) splice site recognition. A classic model involves recognition of splicing enhancers by proteins of the SR family and recognition of splicing silencers by hnRNP proteins. However, proteins of these and other families can promote or inhibit splicing depending upon the location of their binding sites relative to the splice sites and other regulatory sequences. A complex combinatorial interplay between regulatory elements and their cognate factors determines exon definition and regulation. (C) A switch from stabilizing interactions between factors recognizing 3′ and 5′ SS across exons (exon definition) to splice site pairing (intron definition) and tri-snRNP assembly occurs in vertebrate internal exons and can be targeted by regulatory factors like hnRNP L or RBM5. Download figure Download PowerPoint Assembly of spliceosomal complexes onto pre-mRNA follows a stepwise choreography and is supported by at least eight DExD/H-type RNA-dependent ATPases/helicases whose function is either to remodel snRNP composition or to proofread specific transitions along the assembly cycle. The initial step begins with the recognition of the 5′ SS by U1 snRNP and the cooperative binding of the splicing factor 1 (SF1) and of the heterodimer U2AF65/U2AF35 to the BPS region, polypyrimidine tract and 3′ AG, respectively, generating complex E (Fig 2). These molecular interactions then trigger the ATP-dependent recruitment of the U2 snRNP to the BPS region through base-pairing interactions that bulge out the BPS adenosine. U2 snRNP assembly is also assisted by U2 snRNP-associated proteins engaging in RNA–protein interactions with sequences around the BPS region and in protein–protein interactions (e.g., between the SF3B1 protein and U2AF65). Subsequent to complex A formation, the pre-assembled U4-U6-U5 tri-snRNP joins the pre-spliceosome complex to establish complex B. The enzymatic activation of the machinery takes place at this stage through a series of conformational and massive compositional rearrangements (including displacement of U1 and U4 snRNP) to successively form the catalytically active complex B (Bact, B*) and complex C, which host, respectively, the 1st and 2nd trans-esterification reactions of the splicing reaction 1421 (Fig 2). Figure 2. The spliceosome assembly pathwayInitial recognition of the 5′ SS by U1 snRNP and of the 3′ SS region by SF1 (branch point binding protein) and U2AF (complex E) is followed by the ATP-dependent recruitment of U2 snRNP to the branch point region (complex A), concomitant with displacement of SF1. Binding of the U4/5/6 tri-snRNP and the protein-only NineTeen Complex (NTC) leads to formation of complex B, involving also the displacement of proteins present in complex A. Extensive remodeling of complex B, including the destabilization/displacement of U1 and U4 snRNPs, leads to a catalytically active complex (Bact), which upon further conformational rearrangements and changes in protein composition catalyzes the first step of the splicing reaction, leading to the formation of a lariat intermediate containing a 2′-5′ phosphodiester bond (complex C). An additional conformational switch leads to the second catalytic step, rendering the spliced product and the intron lariat. Upon release of the products, the lariat intron is linearized and degraded and the snRNPs recycled for another round of assembly and catalysis in other introns. Proteins of the ATP-dependent DEAH/X helicase family (in magenta) are key to promote the multiple conformational transitions, characterized by extensive rearrangements of RNA:RNA interactions involving snRNA:snRNA and snRNA:pre-mRNA contacts. Download figure Download PowerPoint From constitutive to alternative splicing The core splicing sequences in higher eukaryotes are often variable and contain too little information to unambiguously define SS. Additional sequences in the pre-mRNA modulate SS recognition and are referred to as exonic or intronic splicing enhancers (ESE or ISE) or silencers (ISS or ESS). These sequence elements are recognized by trans-acting splicing factors that balance splice site selection and alternative splicing decisions (Fig 1B). Trans-acting factors include the serine/arginine-rich domain-containing (SR) protein and heterogeneous nuclear ribonucleoprotein (hnRNP) families, which display cooperative or antagonistic effects on the recruitment of the core splicing machinery, typically at early stages of spliceosome formation 22. In addition, several tissue-restricted regulators have been identified, including the neuronal-specific determinants neuro-oncological ventral antigen (NOVA), RNA-binding Fox (RBFOX) or muscleblind (MBNL). Two recurrent themes are that the same factors can act as activators or repressors depending on the position of their binding sites relative to the regulated SS and that their precise activity depends on the context of other cognate sites for other regulatory factors with which they can establish cooperative or antagonistic interactions 23. This leads to a complex interplay of regulatory sequences, positional effects, and trans-acting factor interactions that establish the functional framework of a splicing code 232425. In addition, variations in the levels or activity of core splicing factors, even those acting late in the spliceosome assembly pathway, can also modulate SS choice 182627282930. Furthermore, an increasing number of studies revealed that splicing regulation is also subjected to complex interaction with the transcription and chromatin machineries. Indeed, changes in the kinetics of RNA polymerase II elongation can markedly affect SS selection by influencing the ability of splicing regulators to bind to nascent mRNAs 3132. In addition, histone marks and nucleosome positioning are also key features that participate in splicing reactions by helping the recruitment of splicing regulators and collaborating in exon definition, respectively 13253334. Finally, signal transduction cascades represent another regulatory level through the modulation of posttranslational modifications of splicing regulators, which may modify their interactions, activities, and localization 2335. Alterations of splicing in pathological conditions There is growing evidence from both human genetics and genomewide studies that splicing control can impact a variety of pathologies at three levels (Fig 3): (i) Mutations or genetic variants that affect cis-acting sequences by decreasing the specificity or fidelity of SS selection or activating cryptic SS that are normally not used. These alterations impinge on single genes. (ii) Functional alterations in trans-acting splicing factors, including core spliceosomal components and regulatory factors. Such perturbations potentially modify the expression of multiple RNA targets. (iii) Stoichiometric imbalance of splicing factors following their sequestration in repetitive elements. Such squelching mechanism brings about widespread gene expression changes. While abundant examples of such pathogenic mechanisms exist, it is expected that further combined experimental and computational approaches will greatly expand the repertoire—and possibly the categories of mechanisms—of mutations that determine predisposition, onset and/or progression of pathologies, opening novel opportunities for diagnosis, and translational research. Figure 3. Spectrum of pathologies associated with splicing defectsBroadly classified as mutations in sequence elements (A), alterations in splicing factors (B) and titration/signaling effects of nucleotide repeat expansions (C), the nature of various molecular alterations and their effects on splicing are described for various pathologies associated with splicing defects. Download figure Download PowerPoint Cis-acting mutations: breaking the splicing code Genetic variation within splice site and regulatory sequences frequently causes aberrant splicing in human hereditary diseases and cancer. Single nucleotide substitutions affecting the 5′ or the 3′ SS are the most common splicing mutations, resulting either in exon skipping, activation of a cryptic SS, or to a lesser extent in intron retention. Similarly, intronic mutations and exonic variations (e.g., missense, nonsense, or even otherwise silent mutations) can often trigger splicing perturbations through a loss and/or a gain of enhancers/silencers. This is illustrated by the analysis of the mutational landscape of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene related to cystic fibrosis 36, or of the neurofibromatosis gene, where 50% of the disease-causing mutations lead to splicing defects 37. These alterations can occur in both constitutive and alternative exons and consequently generate aberrant transcripts that miss a constitutive exon or result in changes in the ratio between spliced isoforms (Fig 3A). According to the Human Gene Mutation Database (HGMD release 2014.4), mutations that disrupt normal splicing have been estimated to account for up to a third of all disease-causing mutations 38394041. In silico tools have been developed for predicting the penetrance associated with de novo mutations and the molecular consequences for disease formation 42. These efforts should be complemented with predictions of the impact of frequent intronic regulatory sequences as well as exonic silent mutations (or even missense mutations) that affect splicing outcomes. In fact, synonymous variants have already been demonstrated to contribute to human diseases and be particularly prevalent modulators of exon usage 36434445. Consistent with the observation that cancer-related genes may harbor a greater susceptibility toward aberrant splicing 46, recurrent synonymous mutations in ESE and ESS have been identified for a subset of important oncogenes, representing an extra mechanism for oncogene activation. More globally, half of synonymous drivers is estimated to alter splicing 44, further emphasizing the importance of splicing in cancer biology. The MutPred Splice algorithm aims to determine functional relationships between exonic variants and mis-splicing in inherited diseases and cancer 47. The results indicate that in inherited disease, loss of natural splice sites represents the principal category of splice-altering variants (SAV), whereas ESE loss and/or ESS gain leading to exon skipping is more frequent in cancer 47. More recently, Xiong, Frey, and colleagues assessed the effects of 650,000 single nucleotide intronic and exonic variants using a machine-learning computational pipeline that uncovered an extensive impact of mutation-associated splicing alterations. Their results estimate that intronic mutations alter splicing nine times more frequently than other common variants and that disease-associated missense exonic mutations are five times more likely to interfere with splicing than non-disease-associated variants 45, further illustrating the potential impact of splicing alterations on human pathologies. This approach led to the identification of splicing alterations with potential roles in autism and provided an explanation for the penetrance of synonymous mutations in colorectal cancer 45. The following examples illustrate how disease-causing mutations can be tightly linked to multiple aspects of splice site recognition and, in fact, help to illustrate the delicate balance of sequence signals and interactions that tune splice site choice and can potentially inform therapeutic approaches (see also Figs 3 and 4 and section on therapies). Figure 4. Summary of therapeutic approaches based upon splicing modulationTwo main strategies are outlined. Splice-switching antisense oligonucleotides target splice sites or splicing regulatory sequences to prevent the binding of cognate factors to modulate splice site selection. Examples include blocking of an ISS element that promotes exon 7 inclusion in the SMN2 gene, leading to restoring SMN protein expression and motoneuron function in SMA; induction of skipping of a mutation-containing exon in the DMD gene, leading to in-frame deletion of a nonessential part of the dystrophin protein and restoration of muscle function; prevention of sequestration of the MBNL splicing regulator in CUG repeat expansions in DMPK transcripts, leading to restoration of abnormal splicing patterns in DM. Small molecules, some of them targeting core splicing components like SF3B, modulate alternative splicing of cell cycle control genes and display anti-tumoral properties. Other drugs can induce SMN2 exon 7 inclusion and therefore raise hope as oral treatments for SMA. Download figure Download PowerPoint The vast majority of patients with familial dysautonomia (FD) contain a point mutation at the sixth position of intron 20 of the IKBKAP gene, which encodes the transcription regulator protein IKAP 48. This position is the last of the intronic nucleotides that establish base-pairing interactions with the 5′ end of U1 snRNA as part of the initial step in 5′ SS recognition (Fig 1A). Just the lack of this single base pair leads to defects in 5′ SS identification and in exon definition such that the whole exon 20 is skipped, generating an mRNA with a premature stop codon and defective expression of IKAP 49. The critical role of this single base pair became evident in experimental systems where restoring base-pairing between the mutated 5′ SS and U1 snRNA also restored exon 20 inclusion 50. Remarkably, the extent of the splicing defect is tissue dependent, being very limited in lymphoblasts but extensive in the brain, explaining the severe brain abnormalities and demyelination-associated symptoms of the disease 51. The basis for the tissue-specific effect of the mutation remains obscure. More than 2,500 sequences may function as 5′ SS 52. It is thus not surprising that small sequence variations can lead to the activation of cryptic splice sites, as dramatically illustrated by Hutchinson–Gilford Progeria, a premature aging syndrome where most affected individuals harbor a single C>T silent mutation in exon 11. The mutation activates a cryptic 5′ SS, leading to an mRNA encoding a dominant negative form of lamin A (progerin) that causes nuclear and genomic instability. The balance between the activities of the SR proteins SRSF1 and SRSF6 determines the level of cryptic site activation 53, which can be modulated using antisense oligonucleotides or morpholinos, with therapeutic effects in cell and mouse models of the disease 5455. Strikingly, it has been proposed that even the wild-type sequence can be used as a 5′ SS and that its use increases with age, possibly contributing to physiological aging 54. Another example of how single nucleotide changes near 5′ SS regions can dramatically affect splicing outcomes and disease is tauopathies associated with mutations in exon 10 of the Microtubule Associated Protein Tau (MAPT) gene. These mutations, found in thirteen families with the autosomal dominant condition frontotemporal dementia and parkinsonism linked to chromosome 17 (FTPD-17), alter the ratio between spliced isoforms, promoting exon inclusion and leading to Tau protein aggregation, which has been linked with personality disturbances, dementia, and motor dysfunction 56. The mutations are not located at the splice sites, but rather induce the opening of an RNA stem-loop that normally partially sequesters the 5′ SS, preventing full inclusion of exon 10 5657. This in fact provides one of the best-documented examples of how secondary structures in the pre-mRNA can influence splice site recognition. A now classical example of how sequence variation in exonic sequences can influence exon recognition with profound consequences for human disease is spinal muscular atrophy (SMA). SMA is one of the most frequent genetic diseases, an autosomal recessive neuromuscular disorder characterized by the selective loss of spinal motor neurons, leading to severe skeletal muscle weakness and atrophy. SMA etiology relates to insufficient amount of SMN protein whose function is to chaperone the biogenesis and assembly of snRNPs 12. SMN insufficiency results from loss-of-function mutations or deletion of the Survival Motor Neuron 1 gene (SMN1) 58. Despite its high homology to SMN1, the SMN2 gene fails to prevent SMA development due to a synonymous nucleotide difference, C6T, in exon 7, which causes exon 7 skipping and generation of a truncated, unstable, and rapidly degraded version of the SMN protein 59. Understanding the mechanisms behind the differential effects of C vs. T on exon 7 splicing may indeed be instrumental to offer novel therapeutic approaches because the penetrance of the disease is inversely correlated with the levels of exon 7 inclusion in SMN2, which differ in different patient populations 60. Extensive analyses of the mechanistic impact of the C>T transition initially revealed the loss of an ESE recognized by the SR protein SRSF1 as well as the gain of an ESS recognized by hnRNPA1 6162. Later work revealed additional contributions of intronic silencers that collectively repress exon 7 636465. SMA can be considered as a complex multifactorial pathology as the absence of SMN implies perturbations of the snRNP repertoire with widespread splicing changes 66 and also correlates with splicing alterations of U12 minor spliceosome-dependent events in mouse and Drosophila SMA models 676869. A key and still largely unresol