DS0405 -

Investigating widespread regulation of gene expression through intron retention – WIRED

Submission summary

Alternative splicing affects up to 95% of multi-exonic genes in humans. There are three main types of alternative splicing: exon skipping, alternate 5` or 3` usage and intron retention (IR). While the first two are well described in normal development and disease, the role of IR in these processes remains to be definitively determined. IR occurs when an intron is included in a mature mRNA. Previously regarded as a byproduct of faulty splicing, transcripts with retained introns are often rapidly degraded by a surveillance mechanism called nonsense-mediated decay (NMD). We discovered that normal granulocytic blood cells make use of this mechanism by increasing the amount of transcripts with retained introns of dozens of genes that are essential to granulocytic differentiation (Cell, 2013). We then found a crucial role for IR in the the regulation of pluripotent stem cells (Nature, 2014) and in erythrocyte differentiation (Blood, 2016). Since, we and others have shown that regulation through IR coupled with NMD is evolutionarily conserved, widespread in normal and diseased tissues, and occurs in over 80% of coding genes. Because IR could not previously be correctly identified, numerous studies have overlooked potential biomarkers and therapeutic targets linked to this novel type of gene regulation. The importance of IR in disease is further underlined by the recent finding that tumour suppressor genes are modulated by IR in many different cancers (Nature Genetics, 2015). As such, a greater understanding of regulation through IR could positively impact ongoing and future research in numerous fields that contribute to human health.
In this proposal we will further investigate the molecular mechanisms that lead to IR and develop bioinformatics tools to allow other teams to investigate this mode of regulation in their own data. The model we will use to investigate IR coupled with NMD is that of mouse granulocytic differentiation. These cells can be sorted with over 95% purity and we showed that their fate is orchestrated in part by dramatic changes in IR. They are thus an ideal model to investigate the molecular mechanisms that regulate IR.
We will dissect 2 main mechanisms that cause IR : cis-regulatory motifs that activate or repress IR and epigenetic modifications that change polII elongation rates and thus affect intron recognition by the splicing machinery. To precisely detect motifs that enhance IR, we will first develop a novel approach to more accurately identify and characterize IR events. This approach will combine a third generation sequencing technology (PacBio) that generates ultra-long sequences from single molecules with high throughput Illumina sequencing. From this set of high confidence IR we will detect DNA motifs that are enriched in frequently retained introns and test them using CRISPR-Cas9 genome editing in the MPRO granulocytic cell line. We will then focus on epigenetic changes that may modulate IR. Nucleosome occupancy and DNA methylation through the intermediary of MeCP2 and CTCF proteins can modulate polII elongation and affect mRNA splicing. By assessing nucelosome occupancy, DNA methylation, the binding of MeCP2 and CTCF and the precise identification of IR, we will be able to discover associations between epigenetic marks and IR. We will then test this association by knocking out MeCP2 and CTCF then by altering the methylation status of regions associated with IR using a TALE-Tet1 and a dCas9-DNMT3A system.
This project will develop methods to precisely detect IR events and will allow us to decipher the molecular mechanisms of a this novel mode of gene regulation that has thus far been linked with numerous disorders and normal differentiation processes.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


EE Eduardo Eyras

Help of the ANR 269,163 euros
Beginning and duration of the scientific project: January 2017 - 36 Months

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