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Mechanism of SMN circRNA generation. Splicing factors and U snRNP are recruited during transcription by RNA polymerase II (pol II). Many introns could be potentially removed by forward splicing while pol II is still transcribing. Removal of introns is accompanied by deposition of exon junction complex (EJC) at the exon-exon junction. In some instances, EJC may help recruit splicing factors and promote splicing of neighboring introns. We hypothesize that EJC deposited at the junction of exons 3 and 4 promotes backsplicing involving the 5 ss of exon 4.
Source publication
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 compe...
Contexts in source publication
Context 1
... mechanisms may account for this stimulatory effect, including a better recruitment of U1 snRNP at the 5 ss of exon 4, promoted by cross-exon interactions initiated by factors recruited at the 3 ss of exon 4. Such a mechanism will be consistent with the exon definition model in which recognition of splice sites at each end of an internal exon is promoted by a network of factors enveloping the entire exon [38]. Transcription is coupled to splicing, and removal of intronic sequences is accompanied by the deposition of the exon junction complex, EJC, which is known to promote splicing of the neighboring introns [39,40] (Figure 7). Moreover, eukaryotic initiation factor eIF4A3, one of the components of EJC, has been implicated in circRNA generation [41]. ...
Context 2
... eukaryotic initiation factor eIF4A3, one of the components of EJC, has been implicated in circRNA generation [41]. Hence, it is possible that components of EJC, including eIF4A3, are regulators of SMN circRNA generation (Figure 7). A mutually inclusive hypothesis would be that the intercalating introns increase the size of precursor transcripts, rendering more flexible structures and facilitating backsplicing. ...
Context 3
... results suggest that the structural constraints imposed by the RNA:RNA duplex, which pairs the 5 ss of exon 4 with the 3 ss of an upstream exon, are not conducive for the usage of the alternative (internal) 5 ss generating small circRNAs. Figure 7. Mechanism of SMN circRNA generation. ...
Citations
... To determine the transcriptome-wide effects of overexpression of C2A-2B-3-4, a circRNA harboring SMN1/2 exons 2A, 2B, 3 and 4, we employed the recently reported inducible TC4-2A cell line 44 . For the purposes of comparison, we also employed the inducible TL4-2A cell line that overexpresses L2A-2B-3-4, a linear transcript harboring SMN1/2 exons 2A, 2B, 3 and 4 ( Fig. 2A) 44 . ...
... To determine the transcriptome-wide effects of overexpression of C2A-2B-3-4, a circRNA harboring SMN1/2 exons 2A, 2B, 3 and 4, we employed the recently reported inducible TC4-2A cell line 44 . For the purposes of comparison, we also employed the inducible TL4-2A cell line that overexpresses L2A-2B-3-4, a linear transcript harboring SMN1/2 exons 2A, 2B, 3 and 4 ( Fig. 2A) 44 . As a control for the background expression profile, we used the parental T-REx cell line without any insertion. ...
... Various mechanisms may account for the regulation of genes by C2A-2B-3-4. Previous finding that C2A-2B-3-4 is prominently localized in the cytosol may suggest that the effect of C2A-2B-3-4 on transcriptome is exerted through regulation of translation of transcription factors 44 . This could be achieved through sponging of specific miRNAs and/or sequestration factors associated with translation. ...
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.
... Abbreviations used in this publication are listed in Supplementary Table S1. HEK293-derived stable cell lines TC4-2A and TL4-2A expressing C2A-2B-3-4 and L2A-2B-3-4, receptively, used here were previously generated from the commercially available T-REx cells 44 ...
... Relative expression was determined using the 2 -∆∆Ct method using GAPDH as housekeeping gene. To determine the copy number of circRNA per cell, we used a standard curve de ned by serial dilution of a known quantity of linearized plasmid containing the sequence of the expected PCR product 44 . All primers were obtained from Integrated DNA Technologies. ...
... To determine the transcriptome-wide effect of overexpression of C2A-2B-3-4, a circRNA harboring SMN1/SMN2 exons 2A, 2B, 3 and 4, we employed the recently reported inducible TC4-2A cell line 44 . For the purposes of comparison, we also employed inducible TL4-2A cell line that overexpresses L2A-2B-3-4, a linear transcript harboring SMN1/SMN2 exons 2A, 2B, 3 and 4 ( Fig. 2A) 44 For the remainder of our analyses, we focused on the genes speci cally affected in TC4-2A cells overexpressing C2A-2B-3-4. ...
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 .
... It has been reported that SMN genes produce lots of circRNAs due to the high presence of inverted Alu repeats (Ottesen et al., 2017(Ottesen et al., , 2019Pagliarini et al., 2020). Recently, Luo et al. (2022) found that SMN circRNAs are localized in the cytoplasm. However, the precise function of circRNAs in the pathogenesis of SMA remains unclear. ...
Circular RNAs (circRNAs) are a class of covalently closed single-stranded RNAs that are expressed during the development of specific cells and tissues. CircRNAs play crucial roles in physiological and pathological processes by sponging microRNAs, modulating gene transcription, controlling the activity of certain RNA-binding proteins, and producing functional peptides. A key focus of research at present is the functionality of circRNAs in the nervous system and several advances have emerged over the last 2 years. However, the precise role of circRNAs in the nervous system has yet to be comprehensively reviewed. In this review, we first summarize the recently described roles of circRNAs in brain development, maturity, and aging. Then, we focus on the involvement of circRNAs in various diseases of the central nervous system, such as brain cancer, chronic neurodegenerative diseases, acute injuries of the nervous system, and neuropathic pain. A better understanding of the functionality of circRNAs will help us to develop potential diagnostic, prognostic, and therapeutic strategies to treat diseases of the nervous system.
... The SMN protein was given the name "survival motor neuron" because of the apparent importance of protein to the motor system and the finding that the knockout of SMN in mice was embryonically lethal (Schrank et al., 1997). Recent studies have shown that SMN genes generates a huge repertoire of circRNAs through interintronic secondary structures (Luo et al., 2022;Singh et al., 2022). There are four subtypes of SMN circRNAs namely Type 1, 2, 3 and 4 circRNAs (Ottesen and Singh, 2020). ...
Altered autophagy is a hallmark of neurodegeneration but how autophagy is regulated in the brain and dysfunctional autophagy leads to neuronal death has remained cryptic. Being a key cellular waste-recycling and housekeeping system, autophagy is implicated in a range of brain disorders and altering autophagy flux could be an effective therapeutic strategy and has the potential for clinical applications down the road. Tight regulation of proteins and organelles in order to meet the needs of complex neuronal physiology suggests that there is distinct regulatory pattern of neuronal autophagy as compared to non-neuronal cells and nervous system might have its own separate regulator of autophagy. Evidence has shown that circRNAs participates in the biological processes of autophagosome assembly. The regulatory networks between circRNAs, autophagy, and neurodegeneration remains unknown and warrants further investigation. Understanding the interplay between autophagy, circRNAs and neurodegeneration requires a knowledge of the multiple steps and regulatory interactions involved in the autophagy pathway which might provide a valuable resource for the diagnosis and therapy of neurodegenerative diseases. In this review, we aimed to summarize the latest studies on the role of brain-protective mechanisms of autophagy associated circRNAs in neurodegenerative diseases (including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and Friedreich’s ataxia) and how this knowledge can be leveraged for the development of novel therapeutics against them. Autophagy stimulation might be potential one-size-fits-all therapy for neurodegenerative disease as per considerable body of evidence, therefore future research on brain-protective mechanisms of autophagy associated circRNAs will illuminate an important feature of nervous system biology and will open the door to new approaches for treating neurodegenerative diseases.
