Natalia N. Singh

Iowa State University

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Ravindra Singh

Iowa State University

Eric W. Ottesen

Iowa State University

Joonbae Seo

Iowa State University

Matthew D. Howell

Confederated Tribes of Warm Springs

Elliot J. Androphy

Indiana University – Purdue University Indianapolis

Diou Luo

Iowa State University

Maria Shishimorova

Iowa State University

Brian M. Lee

Coastal Carolina University

Adrian G. Todd

University of Florida

Dhruva Bhattacharya

University of Arizona

Cooperative Institutions

Iowa State University

Howard Hughes Medical Institute

Indiana University – Purdue University Indianapolis

Indiana University School of Medicine

University of Massachusetts Chan Medical School

The University of Texas Health Science Center at Houston

University of Connecticut

Institute of Molecular Biology and Biophysics

Institute of Bioorganic Chemistry, Polish Academy of Sciences

University of Florida

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Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

Methods in molecular biology (2018)

Natalia N. Singh Diou Luo Ravindra Singh

Exonic splicing enhancer

Minigene

10.1007/978-1-4939-8651-4_26

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Citations (38)

TIA1 Prevents Skipping of a Critical Exon Associated with Spinal Muscular Atrophy

Molecular and Cellular Biology (2010)

Natalia N. Singh Joonbae Seo Eric W. Ottesen Maria Shishimorova Dhruva Bhattacharya

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.

Polypyrimidine tract-binding protein

Splicing factor

Polypyrimidine tract

Exon skipping

Minigene

10.1128/mcb.00945-10

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Citations (79)

Splicing regulation in spinal muscular atrophy by an RNA structure formed by long‐distance interactions

Annals of the New York Academy of Sciences (2015)

Natalia N. Singh Brian M. Lee Ravindra Singh

Humans carry two copies of the survival motor neuron gene: SMN1 and SMN2 . Loss of SMN1 coupled with skipping of SMN2 exon 7 causes spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. Our discovery of intronic splicing silencer N1 (ISS‐N1) is a promising target, currently in a phase III clinical trial, for an antisense oligonucleotide–mediated splicing correction in SMA. We have recently shown that the first residue of ISS‐N1 is locked in a unique RNA structure that we term ISTL1 (internal stem through long‐distance interaction–1). Complementary strands of ISTL1 are separated from each other by 279 nucleotides. Using site‐specific mutations and chemical structure probing, we confirmed the formation and functional significance of ISTL1. Located in the middle of intron 7, the 3′ strand of ISTL1 falls within an inhibitory region that we term ISS‐N2. We demonstrate that an antisense oligonucleotide–mediated sequestration of ISS‐N2 fully corrects SMN2 exon 7 splicing and restores high levels of SMN in SMA patient cells. These results underscore the therapeutic potential of the regulatory information present in a secondary and high‐order RNA structure of a human intron.

SMN1

10.1111/nyas.12727

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Citations (52)

RNA in spinal muscular atrophy: therapeutic implications of targeting

Expert Opinion on Therapeutic Targets (2020)

Ravindra Singh Joonbae Seo Natalia N. Singh

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.This review summarizes existing and potential SMA therapies that are based on RNA targeting.We also discuss the mechanistic basis of RNA-targeting molecules.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.

10.1080/14728222.2020.1783241

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Citations (20)

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy

Biochemical and Biophysical Research Communications (2004)

Natalia N. Singh Elliot J. Androphy Ravindra Singh

SMN1

Exonic splicing enhancer

Exon skipping

10.1016/j.bbrc.2004.01.067

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Citations (109)

A survey of transcripts generated by spinal muscular atrophy genes

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms (2020)

Natalia N. Singh Eric W. Ottesen Ravindra Singh

10.1016/j.bbagrm.2020.194562

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Citations (14)

Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene

Genes (2022)

Diou Luo Natalia N. Singh Ravindra Singh

Human survival motor neuron 1 (SMN1) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of SMN1 lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. SMN2, a near identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of SMN2 exon 7. Restoration of SMN by splicing modulation of SMN2 exon 7 or gene replacement are currently approved therapies of SMA. Human SMN genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of SMN circRNA generation has not yet been examined in detail. For example, it remains unknown if forward splicing impacts backsplicing that generates circRNAs containing multiple exons. Here, we employed SMN as a model system to examine the impact of intronic sequences on the generation of circRNAs. We performed our experiments in HeLa cells transiently transfected with minigenes expressing three abundantly represented circRNAs containing two or more SMN exons. We observed an enhanced rate of circRNA generation when introns joining exons to be incorporated into circRNAs were present as compared to the intronless context. These results underscore the stimulatory effect of forward splicing in the generation of circRNAs containing multiple exons. These findings are consistent with the reported low abundance of SMN circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the SMN circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that SMN circRNAs were preferentially localized in the cytoplasm. These findings provide new insights regarding our understanding of circRNA generation and open avenues to uncover novel functions of the SMN genes.

SMN1

Circular RNA

Exon skipping

Minigene

10.3390/genes13071145

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Citations (5)

Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy

PMC (2013)

Sara K. Custer Adrian G. Todd Natalia N. Singh Elliot J. Androphy

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.

Neurite

COPI

SMN1

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Alternative splicing in spinal muscular atrophy underscores the role of an intron definition model

RNA Biology (2011)

Natalia N. Singh Ravindra Singh

absence of a defined upstream 3' ss of an alternatively spliced exon.

10.4161/rna.8.4.16224

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Citations (51)

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

Nucleic Acids Research (2023)

Eric W. Ottesen Natalia N. Singh Diou Luo Bailey Kaas Benjamin Gillette

Abstract 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.

Exon skipping

10.1093/nar/gkad259

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Citations (24)