Abstract
Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5′ splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.
Highlights
Pre-mRNA splicing is an essential step for expression of most eukaryotic genes, during which introns are removed and exons joined to generate a mature mRNA
To systematically explore pentameric and shorter sequences that are critical for splicing regulation, we took advantage of a previously reported SMN2 minigene [30] to generate a complete pentamer library by site-directed mutagenesis, and analyzed the effects of all possible mutants on exon 7 splicing
We mutated the 5-nt sequence CAGCA at positions 11–15 in SMN2 intron 7 into all possible combinations of sequences. We chose this intronic position on the basis of our previous mutational analysis, which showed that suboptimal splicing-regulatory elements (SREs) in this region can noticeably affect exon 7 splicing [15]
Summary
Pre-mRNA splicing is an essential step for expression of most eukaryotic genes, during which introns are removed and exons joined to generate a mature mRNA. The prevalence of pseudo-exons with core signals that match the consensus elements indicates that additional cis-acting splicing signals are involved in distinguishing true exons from pseudo-exons [2,3]. These auxiliary signals, termed splicing-regulatory elements (SREs) represent another essential aspect of the splicing code. Based on their role and position, SREs are classified as exonic and intronic splicing enhancers (ESEs and ISEs) or silencers (ESSs and ISSs). Based on their role and position, SREs are classified as exonic and intronic splicing enhancers (ESEs and ISEs) or silencers (ESSs and ISSs). cis-Acting SREs are typically located in the vicinity of splice sites to exert their effects; distal elements located >500 nt away from
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