A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes.

Here we report a Survival Motor Neuron 2 (SMN2) super minigene, SMN2Sup, encompassing its own promoter, all exons, their flanking intronic sequences and the entire 3'-untranslated region. We confirm that the pre-mRNA generated from SMN2Sup undergoes splicing to produce a translation-competent mRNA. We demonstrate that mRNA generated from SMN2Sup produces more SMN than an identical mRNA generated from a cDNA clone. We uncover that overexpression of SMN triggers skipping of exon 3 of SMN1/SMN2. We define the minimal promoter and regulatory elements associated with the initiation and elongation of transcription of SMN2. The shortened introns within SMN2Sup preserved the ability of camptothecin, a transcription elongation inhibitor, to induce skipping of exons 3 and 7 of SMN2. We show that intron 1-retained transcripts undergo nonsense-mediated decay. We demonstrate that splicing factor SRSF3 and DNA/RNA helicase DHX9 regulate splicing of multiple exons in the context of both SMN2Sup and endogenous SMN1/SMN2. Prevention of SMN2 exon 7 skipping has implications for the treatment of spinal muscular atrophy (SMA). We validate the utility of the super minigene in monitoring SMN levels upon splicing correction. Finally, we demonstrate how the super minigene could be employed to capture the cell type-specific effects of a pathogenic SMN1 mutation.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.

BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.

RNA in spinal muscular atrophy: therapeutic implications of targeting.

INTRODUCTION: Spinal muscular atrophy (SMA) is caused by low levels of the Survival Motor Neuron (SMN) protein due to deletions of or mutations in the SMN1 gene. Humans carry another nearly identical gene, SMN2, which mostly produces a truncated and less stable protein SMNΔ7 due to predominant skipping of exon 7. Elevation of SMN upon correction of SMN2 exon 7 splicing and gene therapy have been proven to be the effective treatment strategies for SMA. AREAS COVERED: This review summarizes existing and potential SMA therapies that are based on RNA targeting.We also discuss the mechanistic basis of RNA-targeting molecules. EXPERT OPINION: The discovery of intronic splicing silencer N1 (ISS-N1) was the first major step towards developing the currently approved antisense-oligonucleotide (ASO)-directed therapy (SpinrazaTM) based on the correction of exon 7 splicing of the endogenous SMN2pre-mRNA. Recently, gene therapy (Zolgensma) has become the second approved treatment for SMA. Small compounds (currently in clinical trials) capable of restoring SMN2 exon 7 inclusion further expand the class of the RNA targeting molecules for SMA therapy. Endogenous RNA targets, such as long non-coding RNAs, circular RNAs, microRNAs and ribonucleoproteins, could be potentially exploited for developing additional SMA therapies.

Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs.

Circular RNAs (circRNAs) perform diverse functions, including the regulation of transcription, translation, peptide synthesis, macromolecular sequestration and trafficking. Inverted Alu repeats capable of forming RNA:RNA duplexes that bring splice sites together for backsplicing are known to facilitate circRNA generation. However, higher limits of circRNAs produced by a single Alu-rich gene are currently not predictable due to limitations of amplification and analyses. Here, using a tailored approach, we report a surprising diversity of exon-containing circRNAs generated by the Alu-rich Survival Motor Neuron (SMN) genes that code for SMN, an essential multifunctional protein in humans. We show that expression of the vast repertoire of SMN circRNAs is universal. Several of the identified circRNAs harbor novel exons derived from both intronic and intergenic sequences. A comparison with mouse Smn circRNAs underscored a clear impact of primate-specific Alu elements on shaping the overall repertoire of human SMN circRNAs. We show the role of DHX9, an RNA helicase, in splicing regulation of several SMN exons that are preferentially incorporated into circRNAs. Our results suggest self- and cross-regulation of biogenesis of various SMN circRNAs. These findings bring a novel perspective towards a better understanding of SMN gene function.

Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology.

RNA silencing inhibits mRNA translation. While mRNA translation accounts for the majority of cellular energy expenditure, it is unclear if RNA silencing regulates energy homeostasis. Here, we report that hepatic Argonaute 2 (Ago2)-mediated RNA silencing regulates both intrinsic energy production and consumption and disturbs energy metabolism in the pathogenesis of obesity. Ago2 regulates expression of specific miRNAs including miR-802, miR-103/107, and miR-148a/152, causing metabolic disruption, while simultaneously suppressing the expression of genes regulating glucose and lipid metabolism, including Hnf1ß, Cav1, and Ampka1. Liver-specific Ago2-deletion enhances mitochondrial oxidation and ATP consumption associated with mRNA translation, which results in AMPK activation, and improves obesity-associated pathophysiology. Notably, hepatic Ago2-deficiency improves glucose metabolism in conditions of insulin receptor antagonist treatment, high-fat diet challenge, and hepatic AMPKα1-deletion. The regulation of energy metabolism by Ago2 provides a novel paradigm in which RNA silencing plays an integral role in determining basal metabolic activity in obesity-associated sequelae.

Cellular Approaches in Investigating Argonaute2-Dependent RNA Silencing.

In mammals, there are four Argonaute (Ago) family proteins that play crucial roles in RNA silencing, a process wherein microRNA (miRNA) mediates inhibition of target mRNA translation. Among the Ago proteins, Argonaute2 (Ago2) uniquely possesses an endoribonuclease (slicer) activity that is critical for the biogenesis of specific miRNAs and mRNA cleavage. This Ago2 slicer activity is required for postnatal development. Despite its important roles, there are still gaps in our understanding of the mechanistic basis of Ago2's unique functions in vivo due to a limited availability of experimental tools. In order to investigate Ago2's functions, we generated a new cellular model of Ago2-deficiency in 3T3 mouse embryonic fibroblasts (MEFs). This cell line can be used for investigating general Ago2 functions, but also for further understanding of Ago2's unique characteristics including the slicer activity, specific amino acid residues, and domains in Ago2 by reconstitution of Ago2 mutants. Here, we describe the methods for establishing Ago2-deficient MEFs and for reconstituting the MEFs with an Ago2 mutant lacking its slicer activity by means of a retrovirus-mediated gene transfer.

A Multilayered Control of the Human Survival Motor Neuron Gene Expression by Alu Elements.

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Mutations or deletions of SMN1, which codes for SMN, cause spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. Aberrant expression or localization of SMN has been also implicated in other pathological conditions, including male infertility, inclusion body myositis, amyotrophic lateral sclerosis and osteoarthritis. SMN2 fails to compensate for the loss of SMN1 due to skipping of exon 7, leading to the production of SMNΔ7, an unstable protein. In addition, SMNΔ7 is less functional due to the lack of a critical C-terminus of the full-length SMN, a multifunctional protein. Alu elements are specific to primates and are generally found within protein coding genes. About 41% of the human SMN gene including promoter region is occupied by more than 60 Alu-like sequences. Here we discuss how such an abundance of Alu-like sequences may contribute toward SMA pathogenesis. We describe the likely impact of Alu elements on expression of SMN. We have recently identified a novel exon 6B, created by exonization of an Alu-element located within SMN intron 6. Irrespective of the exon 7 inclusion or skipping, transcripts harboring exon 6B code for the same SMN6B protein that has altered C-terminus compared to the full-length SMN. We have demonstrated that SMN6B is more stable than SMNΔ7 and likely functions similarly to the full-length SMN. We discuss the possible mechanism(s) of regulation of SMN exon 6B splicing and potential consequences of the generation of exon 6B-containing transcripts.

TIA1 is a gender-specific disease modifier of a mild mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. The nearly identical SMN2 cannot compensate for SMN1 loss due to exon 7 skipping. The allele C (C +/+) mouse recapitulates a mild SMA-like phenotype and offers an ideal system to monitor the role of disease-modifying factors over a long time. T-cell-restricted intracellular antigen 1 (TIA1) regulates SMN exon 7 splicing. TIA1 is reported to be downregulated in obese patients, although it is not known if the effect is gender-specific. We show that female Tia1-knockout (Tia1 -/-) mice gain significant body weight (BW) during early postnatal development. We next examined the effect of Tia1 deletion in novel C +/+/Tia1 -/- mice. Underscoring the opposing effects of Tia1 deletion and low SMN level on BW gain, both C +/+ and C +/+/Tia1 -/- females showed similar BW gain trajectory at all time points during our study. We observed early tail necrosis in C +/+/Tia1 -/- females but not in males. We show enhanced impairment of male reproductive organ development and exacerbation of the C +/+/Tia1 -/- testis transcriptome. Our findings implicate a protein factor as a gender-specific modifier of a mild mouse model of SMA.

A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein.

Spinal muscular atrophy (SMA), a leading genetic disease of children and infants, is caused by mutations or deletions of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to skipping of exon 7. SMN2 predominantly produces SMNΔ7, an unstable protein. Here we report exon 6B, a novel exon, generated by exonization of an intronic Alu-like sequence of SMN. We validate the expression of exon 6B-containing transcripts SMN6B and SMN6BΔ7 in human tissues and cell lines. We confirm generation of SMN6B transcripts from both SMN1 and SMN2. We detect expression of SMN6B protein using antibodies raised against a unique polypeptide encoded by exon 6B. We analyze RNA-Seq data to show that hnRNP C is a potential regulator of SMN6B expression and demonstrate that SMN6B is a substrate of nonsense-mediated decay. We show interaction of SMN6B with Gemin2, a critical SMN-interacting protein. We demonstrate that SMN6B is more stable than SMNΔ7 and localizes to both the nucleus and the cytoplasm. Our finding expands the diversity of transcripts generated from human SMN genes and reveals a novel protein isoform predicted to be stably expressed during conditions of stress.

Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene.

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein.

Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN), a multifunctional protein essential for higher eukaryotes. While SMN is one of the most scrutinized proteins associated with neurodegeneration, its gender-specific role in vertebrates remains unknown. We utilized a mild SMA model (C/C model) to examine the impact of low SMN on growth and development of mammalian sex organs. We show impaired testis development, degenerated seminiferous tubules, reduced sperm count and low fertility in C/C males, but no overt sex organ phenotype in C/C females. Underscoring an increased requirement for SMN expression, wild type testis showed extremely high levels of SMN protein compared to other tissues. Our results revealed severe perturbations in pathways critical to C/C male reproductive organ development and function, including steroid biosynthesis, apoptosis, and spermatogenesis. Consistent with enhanced apoptosis in seminiferous tubules of C/C testes, we recorded a drastic increase in cells with DNA fragmentation. SMN was expressed at high levels in adult C/C testis due to an adult-specific splicing switch, but could not compensate for low levels during early testicular development. Our findings uncover novel hallmarks of SMA disease progression and link SMN to general male infertility.

A short antisense oligonucleotide ameliorates symptoms of severe mouse models of spinal muscular atrophy.

Recent reports underscore the unparalleled potential of antisense-oligonucleotide (ASO)-based approaches to ameliorate various pathological conditions. However, in vivo studies validating the effectiveness of a short ASO (<10-mer) in the context of a human disease have not been performed. One disease with proven amenability to ASO-based therapy is spinal muscular atrophy (SMA). SMA is a neuromuscular disease caused by loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Correction of aberrant splicing of the remaining paralog, SMN2, can rescue mouse models of SMA. Here, we report the therapeutic efficacy of an 8-mer ASO (3UP8i) in two severe models of SMA. While 3UP8i modestly improved survival and function in the more severe Taiwanese SMA model, it dramatically increased survival, improved neuromuscular junction pathology, and tempered cardiac deficits in a new, less severe model of SMA. Our results expand the repertoire of ASO-based compounds for SMA therapy, and for the first time, demonstrate the in vivo efficacy of a short ASO in the context of a human disease.

Antisense methods to modulate pre-mRNA splicing.

The dynamic process of pre-mRNA splicing is regulated by combinatorial control exerted by overlapping cis-elements that are unique to every exon and its flanking intronic sequences. Splicing cis-elements are usually 4-8-nucleotide-long linear motifs that furnish interaction sites for specific proteins. Secondary and higher-order RNA structures exert an additional layer of control by providing accessibility to cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate alternative splicing in vivo. Consistently, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of aberrant splicing in pathological conditions. Here we describe the application of an ASO-based approach for the enhanced production of the full-length mRNA of SMN2 in spinal muscular atrophy patient cells.

Spinal muscular atrophy: an update on therapeutic progress.

Humans have two nearly identical copies of survival motor neuron gene: SMN1 and SMN2. Deletion or mutation of SMN1 combined with the inability of SMN2 to compensate for the loss of SMN1 results in spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. SMA affects 1 in ~6000 live births, a frequency much higher than in several genetic diseases. The major known defect of SMN2 is the predominant exon 7 skipping that leads to production of a truncated protein (SMNΔ7), which is unstable. Therefore, SMA has emerged as a model genetic disorder in which almost the entire disease population could be linked to the aberrant splicing of a single exon (i.e. SMN2 exon 7). Diverse treatment strategies aimed at improving the function of SMN2 have been envisioned. These strategies include, but are not limited to, manipulation of transcription, correction of aberrant splicing and stabilization of mRNA, SMN and SMNΔ7. This review summarizes up to date progress and promise of various in vivo studies reported for the treatment of SMA.

A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes.

Humans have two near identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 coupled with the predominant skipping of SMN2 exon 7 causes spinal muscular atrophy (SMA), a neurodegenerative disease. SMA patient cells devoid of SMN1 provide a powerful system to examine splicing pattern of various SMN2 exons. Until now, similar system to examine splicing of SMN1 exons was unavailable. We have recently screened several patient cell lines derived from various diseases, including SMA, Alzheimer's disease, Parkinson's disease and Batten disease. Here we report a Batten disease cell line that lacks functional SMN2, as an ideal system to examine pre-mRNA splicing of SMN1. We employ a multiple-exon-skipping detection assay (MESDA) to capture simultaneously skipping of multiple exons. Our results show surprising diversity of splice isoforms and reveal novel splicing events that include skipping of exon 4 and co-skipping of three adjacent exons of SMN. Contrary to the general belief, MESDA captured oxidative-stress induced skipping of SMN1 exon 5 in several cell types, including non-neuronal cells. We further demonstrate that the predominant SMN2 exon 7 skipping induced by oxidative stress is modulated by a combinatorial control that includes promoter sequence, endogenous context, and the weak splice sites. We also show that an 8-mer antisense oligonucleotide blocking a recently described GC-rich sequence prevents SMN2 exon 7 skipping under the conditions of oxidative stress. Our findings bring new insight into splicing regulation of an essential housekeeping gene linked to neurodegeneration and infant mortality.

TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy.

Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.