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A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes.
- Source :
-
Nucleic acids research [Nucleic Acids Res] 2024 Apr 24; Vol. 52 (7), pp. 3547-3571. - Publication Year :
- 2024
-
Abstract
- 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.<br /> (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Subjects :
- Humans
RNA Splicing
Serine-Arginine Splicing Factors metabolism
Serine-Arginine Splicing Factors genetics
Nonsense Mediated mRNA Decay
RNA, Messenger genetics
RNA, Messenger metabolism
DEAD-box RNA Helicases genetics
DEAD-box RNA Helicases metabolism
RNA Precursors metabolism
RNA Precursors genetics
Survival of Motor Neuron 2 Protein genetics
Survival of Motor Neuron 2 Protein metabolism
Introns genetics
Survival of Motor Neuron 1 Protein genetics
Survival of Motor Neuron 1 Protein metabolism
Promoter Regions, Genetic
Exons
Transcription, Genetic
Subjects
Details
- Language :
- English
- ISSN :
- 1362-4962
- Volume :
- 52
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Nucleic acids research
- Publication Type :
- Academic Journal
- Accession number :
- 38214229
- Full Text :
- https://doi.org/10.1093/nar/gkad1259