Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient

Ellis, Joseph A.; Hale, Melissa A.; Cleary, John D.; Wang, Eric; Berglund, J. Andrew

doi:10.1016/j.jmb.2023.168156

Cited by 7 publications

(3 citation statements)

References 59 publications

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“…S4A ). Exon sequencing read counts are, however, dependent on the PSI measured in HEK293 cells ( Ellis et al 2023 ; Supplemental Fig. S4B ), and alternative HEK293 mRNA exons are frequently missing in the exon trapping data ( Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

The human genome contains over a million autonomous exons

Stepankiw,

Yang,

Hughes

2023

Genome Res.

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Mammalian mRNA and lncRNA exons are often small compared to introns. The exon definition model predicts that exons splice autonomously, dependent on proximal exon sequence features, explaining their delineation within large introns. This model has not been examined on a genome-wide scale, however, leaving open the question of how often mRNA and lncRNA exons are autonomous. It is also unknown how frequently such exons can arise by chance. Here, we directly assayed large fragments (500–1000 bp) of the human genome by exon trapping, which detects exons spliced into a heterologous transgene, here designed with a large intron context. We define the trapped exons as “autonomous.” We obtained ∼1.25 million trapped exons, including most known mRNA and well-annotated lncRNA internal exons, demonstrating that human exons are predominantly autonomous. mRNA exons are trapped with the highest efficiency. Nearly a million of the trapped exons are unannotated, most located in intergenic regions and antisense to mRNA, with depletion from the forward strand of introns. These exons are not conserved, suggesting they are nonfunctional and arose from random mutations. They are nonetheless highly enriched with known splicing promoting sequence features that delineate known exons. Novel autonomous exons are more numerous than annotated lncRNA exons, and computational models also indicate they will occur with similar frequency in any randomly generated sequence. These results show that most human coding exons splice autonomously, and provide an explanation for the existence of many unconserved lncRNAs, as well as a new annotation and inclusion levels of spliceable loci in the human genome.

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Section: Resultsmentioning

confidence: 99%

The human genome contains over a million autonomous exons

Stepankiw,

Yang,

Hughes

2023

Genome Res.

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“…Total RNA from HA-MBNL1 and ddHEK cells was isolated using the RNeasy Mini kit (Qiagen, Germantown, MD, USA). The isolated RNA was processed via reverse-transcription (RT)-PCR, and PSI (percent spliced in) for each minigene event was calculated as previously described (17). 1000 ng of RNA was reverse-transcribed (or 500 ng for the ASO protocol) using SuperScript IV (Invitrogen, Carlsbad, CA, USA) with random hexamer priming (Integrated DNA Technologies, Coralville, IA, USA) according to the manufacturer's protocol, except that half of the recommended SuperScript IV enzyme was utilized.…”

Section: Cell-based Splicing Assays and Curve Fittingmentioning

confidence: 99%

“…MBNL proteins have been shown to bind to YGCY cis-regulatory motifs to regulate AS outcomes of their target pre-mRNAs in a positional-dependent manner (12,13). Specifically, MBNL proteins generally promote cassette exon inclusion by binding to downstream RNA motifs and repressing or blocking exon inclusion by binding upstream intronic or exonic elements (14)(15)(16)(17). In addition to MBNL proteins, other RBPs such as NOVA, RBFOX, and CELF exhibit positional-dependent patterns in regulating AS outcomes (18)(19)(20)(21)(22)(23)(24)(25)(26).…”

Section: Introductionmentioning

confidence: 99%

Mutation of two intronic nucleotides alters RNA structure and dynamics inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

Nowzari,

Hale,

Ellis

et al. 2024

Preprint

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Alternative splicing (AS) of Exon 11 of the Insulin Receptor (INSR) is highly regulated and disrupted in several human disorders. To better understandINSRexon 11 AS regulation, splicing activity of anINSRexon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA.Abstract figure:Model forINSRexon 11 splicing regulation through the UGCAUG motif. We propose that the UGCAUG motif, MBNL1, and RBFOX1 cooperatively regulateINSRexon 11 splicing. Mutating this UGCAUG motif is sufficient to alter RNA structural dynamics to disrupt this regulation.

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RNA binding proteins in cardiovascular development and disease

Verma,

Kuyumcu-Martinez

2024

Current Topics in Developmental Biology

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Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient

Cited by 7 publications

References 59 publications

The human genome contains over a million autonomous exons

The human genome contains over a million autonomous exons

Mutation of two intronic nucleotides alters RNA structure and dynamics inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

RNA binding proteins in cardiovascular development and disease

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