Deficiency of U4atac snRNA secondarily results in ciliary defects

In the human genome, about 700 genes contain usually one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its non-coding gene, RNU4ATAC, has been found mutated in Taybi-Linder (MOPD1/TALS), Roifman (RFMN) and Lowry-Wood syndromes (LWS). These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy and immunodeficiency. Here, we report a homozygous RNU4ATAC mutation in the Stem II domain, n.16G>A, in two unrelated patients presenting with both typical traits of the Joubert syndrome (JBTS), a well-characterized ciliopathy, and of TALS/RFMN/LWS, thus widening the clinical spectrum of RNU4ATAC-associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. This finding is supported by alterations of primary cilium function in TALS and JBTS/RFMN fibroblasts, as well as by u4atac zebrafish model, which exhibit ciliopathy-related phenotypes and ciliary defects. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.

Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and GABA-A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.

RNA Targeting in Inherited Neuromuscular Disorders: Novel Therapeutic Strategies to Counteract Mis-Splicing

Neuromuscular disorders represent multifaceted abnormal conditions, with little or no cure, leading to patient deaths from complete muscle wasting and atrophy. Despite strong efforts in the past decades, development of effective treatments is still urgently needed. Advent of next-generation sequencing technologies has allowed identification of novel genes and mutations associated with neuromuscular pathologies, highlighting splicing defects as essential players. Deciphering the significance and relative contributions of defective RNA metabolism will be instrumental to address and counteract these malignancies. We review here recent progress on the role played by alternative splicing in ensuring functional neuromuscular junctions (NMJs), and its involvement in the pathogenesis of NMJ-related neuromuscular disorders, with particular emphasis on congenital myasthenic syndromes and muscular dystrophies. We will also discuss novel strategies based on oligonucleotides designed to bind their cognate sequences in the RNA or targeting intermediary of mRNA metabolism. These efforts resulted in several chemical classes of RNA molecules that have recently proven to be clinically effective, more potent and better tolerated than previous strategies.

Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

Intermixing the OPN1LW and OPN1MW Genes Disrupts the Exonic Splicing Code Causing an Array of Vision Disorders

Light absorption by photopigment molecules expressed in the photoreceptors in the retina is the first step in seeing. Two types of photoreceptors in the human retina are responsible for image formation: rods, and cones. Except at very low light levels when rods are active, all vision is based on cones. Cones mediate high acuity vision and color vision. Furthermore, they are critically important in the visual feedback mechanism that regulates refractive development of the eye during childhood. The human retina contains a mosaic of three cone types, short-wavelength (S), long-wavelength (L), and middle-wavelength (M) sensitive; however, the vast majority (~94%) are L and M cones. The OPN1LW and OPN1MW genes, located on the X-chromosome at Xq28, encode the protein component of the light-sensitive photopigments expressed in the L and M cones. Diverse haplotypes of exon 3 of the OPN1LW and OPN1MW genes arose thru unequal recombination mechanisms that have intermixed the genes. A subset of the haplotypes causes exon 3- skipping during pre-messenger RNA splicing and are associated with vision disorders. Here, we review the mechanism by which splicing defects in these genes cause vision disorders.

Intermixing the OPN1LW and OPN1MW Genes Disrupts the Exonic Splicing Code Causing an Array of Vision Disorders

The first step in seeing is light absorption by photopigment molecules expressed in the photore-ceptors of the retina. There are two types of photoreceptors in the human retina that are respon-sible for image formation, rods and cones. Except at very low light levels when rods are active, all vision is based on cones. Cones mediate high acuity vision and color vision. Furthermore, they are critically important in the visual feedback mechanism that regulates refractive development of the eye during childhood. The human retina contains a mosaic of three cone types, short-wavelength (S), long-wavelength (L) and middle-wavelength (M); however, the vast major-ity (~94%) are L and M cones. The OPN1LW and OPN1MW genes, located on the X-chromosome at Xq28, encode the protein component of the light-sensitive photopigments. Here we review mechanism by which splicing defects in these genes cause vision disorders.

Emerging roles of spliceosome in cancer and immunity

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by an intricate ribonucleoprotein complex called the spliceosome. Although the spliceosome is considered to be general cell “housekeeping” machinery, mutations in core components of the spliceosome frequently correlate with cell- or tissue-specific phenotypes and diseases. In this review, we expound the links between spliceosome mutations, aberrant splicing, and human cancers. Remarkably, spliceosome-targeted therapies (STTs) have become efficient anti-cancer strategies for cancer patients with splicing defects. We also highlight the links between spliceosome and immune signaling. Recent studies have shown that some spliceosome gene mutations can result in immune dysregulation and notable phenotypes due to mis-splicing of immune-related genes. Furthermore, several core spliceosome components harbor splicing-independent immune functions within the cell, expanding the functional repertoire of these diverse proteins.

Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation.

Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.