Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene
Human <i>survival motor neuron 1</i> (<i>SMN1</i>) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of <i>SMN1</i> lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease lin...
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MDPI AG
2022-06-01
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| Series: | Genes |
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| author | Diou Luo Natalia Nikolaevna Singh Ravindra Narayan Singh |
| author_facet | Diou Luo Natalia Nikolaevna Singh Ravindra Narayan Singh |
| author_sort | Diou Luo |
| collection | DOAJ |
| description | Human <i>survival motor neuron 1</i> (<i>SMN1</i>) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of <i>SMN1</i> lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. <i>SMN2</i>, a near identical copy of <i>SMN1</i> present in humans, cannot compensate for the loss of <i>SMN1</i> due to predominant skipping of <i>SMN2</i> exon 7. Restoration of SMN by splicing modulation of <i>SMN2</i> exon 7 or gene replacement are currently approved therapies of SMA. Human <i>SMN</i> genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of <i>SMN</i> 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 <i>SMN</i> 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 <i>SMN</i> 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 <i>SMN</i> circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the <i>SMN</i> circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that <i>SMN</i> 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 <i>SMN</i> genes. |
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| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-09T10:17:57Z |
| publishDate | 2022-06-01 |
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| spelling | doaj.art-1118d3443ef14347868b600802566c7e2023-12-01T22:11:20ZengMDPI AGGenes2073-44252022-06-01137114510.3390/genes13071145Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy GeneDiou Luo0Natalia Nikolaevna Singh1Ravindra Narayan Singh2Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USADepartment of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USADepartment of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USAHuman <i>survival motor neuron 1</i> (<i>SMN1</i>) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of <i>SMN1</i> lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. <i>SMN2</i>, a near identical copy of <i>SMN1</i> present in humans, cannot compensate for the loss of <i>SMN1</i> due to predominant skipping of <i>SMN2</i> exon 7. Restoration of SMN by splicing modulation of <i>SMN2</i> exon 7 or gene replacement are currently approved therapies of SMA. Human <i>SMN</i> genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of <i>SMN</i> 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 <i>SMN</i> 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 <i>SMN</i> 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 <i>SMN</i> circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the <i>SMN</i> circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that <i>SMN</i> 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 <i>SMN</i> genes.https://www.mdpi.com/2073-4425/13/7/1145spinal muscular atrophySMAsurvival motor neuronSMNcircular RNAcircRNA |
| spellingShingle | Diou Luo Natalia Nikolaevna Singh Ravindra Narayan Singh Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene Genes spinal muscular atrophy SMA survival motor neuron SMN circular RNA circRNA |
| title | Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene |
| title_full | Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene |
| title_fullStr | Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene |
| title_full_unstemmed | Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene |
| title_short | Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene |
| title_sort | internal introns promote backsplicing to generate circular rnas from spinal muscular atrophy gene |
| topic | spinal muscular atrophy SMA survival motor neuron SMN circular RNA circRNA |
| url | https://www.mdpi.com/2073-4425/13/7/1145 |
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