High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA–SMN interactions. Our results reveal that motifs that drive RNA–SMN interactions are diverse and suggest that tight RNA–SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA– SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions

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

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.This article is published as Luo, Diou, Natalia Nikolaevna Singh, and Ravindra Narayan Singh. "Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene." Genes 13, no. 7 (2022): 1145. DOI: 10.3390/genes13071145. Copyright 2022 by the authors. Attribution 4.0 International (CC BY 4.0). Posted with permission

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 ofSMN 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

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2, produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4,172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/SMN2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/SMN2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/SMN2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, a universally expressed circRNA produced by SMN1/SMN2.This is a preprint from Luo, Diou, Eric Ottesen, Ji Heon Lee, and Ravindra Singh. "Transcriptome-and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes." (2024). doi: https://doi.org/10.21203/rs.3.rs-3818622/v1. Copyright 2024 The Authors. This work is licensed under a CC BY 4.0 License

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A–2B–3–4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A–2B–3–4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A–2B–3–4. About half of the affected genes by C2A–2B–3–4 remained unaffected by L2A–2B–3–4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A–2B–3–4 in gene regulation. A surprisingly high number of upregulated genes by C2A–2B–3–4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A–2B–3–4 and L2A–2B–3–4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A–2B–3–4 with very limited overlap with those affected by L2A–2B–3–4. Independent validations confirmed the effect of C2A–2B–3–4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A–2B–3–4, and expands our understanding of functions of SMN1/2 genes.This article is published as Luo, Diou, Eric W. Ottesen, Ji Heon Lee, and Ravindra N. Singh. "Transcriptome-and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes." Scientific Reports 14, no. 1 (2024): 10442. doi: https://doi.org/10.1038/s41598-024-60593-7

Activation of a cryptic 5′ splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene

Spinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5′ splice site (5′ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5′ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3′ss and 5′ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA

Veterinary Considerations for the Theoretical Resurrection of Extinct Species

The de-extinction of the dinosaur is a dubious possibility but its consideration brings forth some issues that are at least worthy of scientific discussion. In this review, we discuss two distinct issues that have implications for a de-extinct species such as a dinosaur: the ability, or lack thereof, to safely sedate a rare and potentially fractious animal capable of harming the veterinary staff tasked with its care; and, disease risks associated with a species that has been extinct for millions of years. To identify potential sedatives, comparative pharmacology will be needed to uncover the links between receptor pharmacology and the desired clinical outcomes of activating established alpha-2 adrenergic, opioid, and benzodiazepine receptors. Specific to disease control, it will be necessary to understand the unique susceptibility of the new species to current diseases as well as predicting their reservoir capacity for potential human and veterinary pandemic diseases. While the topics presented herein are not exhaustive, this review highlights some of the foremost research that should be conducted in order to serve the unique veterinary needs of a de-extinct species using the dinosaur as a paradigm. Addressing these issues should be considered if an intact dinosaur genome becomes available, regardless of the feasibility of dinosaur resurrection

Theoretical Engineering of the Gut Micro biome for the Purpose of Creating Superior Soldiers

The purpose of this review is to highlight research raising the possibility of exploiting the host-microbiome gut axis for military purposes. Through optimizing the gut-microbiome environment it is possible to enhance nutritional access to indigestible material, provide local and systemic analgesia, enhance psychological robustness to battlefield stress, produce endogenous steroids, reduce muscle fatigue, and promote peripheral wound healing. However, this approach is still in its early stages and thus has not been explored to its full potential. The challenges that are currently preventing the practical use of gut bacteria include the following: inconsistency of clinical outcomes, transient effects requiring continuous supplementation, the type of regimen selected, the initiation and cessation of regimen, and the broader clinical studies needed to validate this research. This review is intended to shed light on the numerous and varied positive impacts such an approach could have for the military if further developed

Characterization of circular RNAs of Survival Motor Neuron genes

Spinal Muscular Atrophy (SMA) is a destructive genetic disease associated with infant and child mortality with a carrier frequency of ~1 in 51 and affecting ~1 in 10,000 live births. Deficiency of the Survival Motor Neuron (SMN) protein owing to homologous disruption or deletion of the SMN1 gene accounts for more than ~90% of SMA cases. So far, most functional studies of the SMN genes are channeled to the SMN protein and its isoforms. However, limited information has been explored regarding SMN transcripts. This study first identified a broad spectrum of circular RNAs (circRNAs) generated by SMN genes involving all the annotated exons by using a tailored approach highlighted with RNase R treatment and PCR with divergent primers. We confirmed our findings in both cells and/or tissues from humans and mice. The result implied that DHX9, an RNA helicase, plays a role in regulating several SMN circRNAs. This study also showed the cross-regulation of SMN circRNA incorporated early exons. And still, the mechanism of SMN circRNA biogenesis remains largely unexplored. Next, constructs and stable cell lines were generated to overexpress three of the most abundantly expressed SMN circRNAs and their linear counterparts without or with internal introns. These circRNAs are C2A-2B-3-4, C2B-3-4 and C3-4, formed by the first four internal exons of SMN. When intercalating introns were encompassed in the constructs, augmented circRNA expression levels were perceived compared to intronless ones, suggesting a stimulatory effect of internal introns on backsplicing to generate SMN circRNAs. The findings also showed that SMN circRNAs were primarily localized in the cytoplasm. Subsequently, I am interested in uncovering the functional roles of SMN circRNAs. Through mechanisms including sponging/decoying miRNAs, interaction with RNA-binding proteins, and serving as translation templates, circRNAs regulate protein expressions and activities. Finally, the proteomics profile of the circRNA-overexpression cell lines was investigated via label-free LC-MS/MS quantification and bioinformatics analysis and validated our findings via qPCR and western blot. We identified several lead proteins associated with neuropathology, neurodevelopment, tumorigenesis, and spermatogenesis. These findings revealed the remarkable diversity of SMN circRNAs and provided pivotal insight into understanding the mechanism of their biogenesis and functions. This study opens novel avenues to exploit SMN-related applications, even beyond the scope of SMA

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

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