A Dynamic Intron Retention Program in the Mammalian Megakaryocyte and Erythrocyte Lineages

Abstract

Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing. Disclosures No relevant conflicts of interest to declare.

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

“…This exon was expressed in erythroblasts as a 58-200nt cassette, depending on alternative splice site usage (Figures S2 and S3). Further inspection of splice junction reads revealed candidate decoy cassettes in retained introns identified earlier in DDX39B, SPTA1, KEL, SNRNP70, and FUS transcripts ((Edwards et al 2016; Pimentel et al 2016); Figure S3). In each case, the putative decoy is flanked on both sides by retained intron(s).…”

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

“…At a constant transcription rate, greater diversion of pre-mRNA into untranslated IR-transcripts should reduce output of mRNA and decrease protein synthesis. Coordinate regulation of IR can effect programmed changes in gene expression patterns during normal development as cells differentiate and respond to environmental signals (Boutz et al 2015;Ni et al 2016;Wong et al 2013;Mauger et al 2016;Braun et al 2017;Naro et al 2017;Edwards et al 2016;Pimentel et al 2016;Shalgi et al 2014). Conversely, aberrations in the IR program are observed in many diseases including cancers where they can adversely impact expression of many genes (Dvinge and Bradley 2015;Luisier et al 2018;Adusumalli et al 2019).…”

“…Analysis of RNA-seq profiles from differentiating erythroid cell populations revealed highly dynamic, global changes in the erythroid transcriptome, including changes in RNA processing of both cassette exons and retained introns, as the cells undergo extensive remodeling during the final cell divisions prior to enucleation Pimentel et al 2014;Pimentel et al 2016;Edwards et al 2016). The IR program encompasses hundreds of IR-transcripts that are polyadenylated and spliced except for selective retention of one or more introns (Pimentel et al 2016;Edwards et al 2016). In late erythroblasts, numerous IR transcripts are abundantlyexpressed, many of which comprise ≥25% of the steady state RNA from their cognate genes.…”

The fitness cost of mis-splicing is the main determinant of alternative splicing patterns

“…This pattern is widespread in eukaryotes and is not restricted to genes encoding splicing factors. Importantly, patterns of AS vary among tissues and during cell differentiation (Wong et al 2013;Braunschweig et al 2014;Edwards et al 2016). This led several authors to propose that AS-NMD might play a critical role in broadly 90 regulating expression of a large percentage of genes (Wong et al 2013;Braunschweig et al 2014;Ge and Porse 2014;Smith and Baker 2015;Wong et al 2016;Edwards et al 2016).…”