Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment

Wong, Justin; Gao, Dadi; Nguyen, Trung Viet; Kwok, Chau-To; Geldermalsen, Michelle van; Middleton, Rob; Pinello, Natalia; Thoeng, Annora; Nagarajah, Rajini; Holst, Jeff; Ritchie, William; Rasko, John E.J.

doi:10.1038/ncomms15134

Cited by 101 publications

(116 citation statements)

References 63 publications

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“…Changing the GC content in and around retained introns had only the potential to repress, but not to enhance splicing ( Fig S2H). A differential effect of CG vs GC dinucleotides in the exon could be observed also here ( Fig 2H; p=9.0x10 -11 , Wilcoxon signed-rank test), supporting recent observations showing a relationship between loss of DNA methylation and increased intron retention (Kim et al, 2018;Wong et al, 2017).…”

Section: Resultssupporting

confidence: 91%

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Mikl

Hamburg

Pilpel

et al. 2018

Preprint

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Most human genes are alternatively spliced, allowing for a large expansion of the proteome. The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematic sequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate both cause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75-0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel, single-cell perspective on splicing regulation.

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Section: Resultssupporting

confidence: 91%

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Mikl

Hamburg

Pilpel

et al. 2018

Preprint

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“…Interestingly, for two genes (SF3B1 and SPTA1), blocking the decoy exon essentially eliminated IR, suggesting that the decoy pathway may be the sole determinant of IR. In contrast, IR was not completely abrogated by antisense treatment in the OGT gene, consistent with the co-existence of decoy-independent IR mechanisms (Monteuuis et al 2019;Braun et al 2017;Cho et al 2014;Wong et al 2017). Finally, in the one case tested, OGT, decreased IR was accompanied by increases in spliced RNA and protein expression.…”

Section: Discussionmentioning

confidence: 59%

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts

Parra

Zhang

et al. 2020

Preprint

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AbstractThe decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides blocking decoy splice sites in endogenous pre-mRNA should increase productive gene expression by reducing IR. Indeed, we now demonstrate that targeting a decoy 5′ splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since IR was nearly eliminated by antisense targeting of 5′ splice sites. Genes in the latter group encode the widely expressed splicing factor (SF3B1), and the erythroid-specific structural protein, alpha-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR, and suggest that dynamic regulation of decoy exons could be a mechanism to fine tune gene expression post-transcriptionally in many cell types.

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“…We anticipate that analogous subsets of IR events might be regulated by decoy mechanisms in other developmental or physiological contexts. Possible candidates could include differentiating granulocytes (Wong et al 2013; Wong et al 2017), activated T cells (Ni et al 2016), stimulated neurons (Mauger et al 2016), cells subjected to proteotoxic stress (Shalgi et al 2014), proliferating vs differentiated muscle cells (Llorian et al 2016), differentiating germ cells (Naro et al 2017), etc. Identifying the RBPs that regulate these decoy-dependent programs will be an important goal of future studies in this area.…”

Section: Discussionmentioning

confidence: 99%

“…How these sub-programs are regulated is not well understood. Recent studies have implicated transcription rate/pausing (Braunschweig et al 2014), specific splicing factors (SRSF4 and HNRNPLL), and DNA and protein methylation factors (by MECP2, in granulocytes, and by PRMT5, in glioblastoma) as important effectors of IR (Cho et al 2014; Boutz et al 2015; Braun et al 2017; Wong et al 2017). In the brain, signaling-dependent splicing was observed to require NMDA-type glutamate receptor or calmodulin-dependent protein kinase pathways for removal of retained introns upon neuronal activation (Mauger et al 2016).…”

Section: Introductionmentioning

confidence: 99%

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

Parra¹,

Booth²,

Weiszmann³

et al. 2018

Preprint

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During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed splice junctions connecting constitutive exons 4 and 5 to cryptic cassette exons encoded in highly conserved intron sequences. In minigene splicing reporter assays, these cassette exons functioned to promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1kb) retained introns than they are in small retained introns or in non-retained introns. Functional assays showed that heterologous cassettes could promote retention of intron 4 in the SF3B1 splicing reporter. Although most of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

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Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment

Cited by 101 publications

References 63 publications

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

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