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A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages - PubMed

  • ️Fri Jan 01 2016

A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages

Christopher R Edwards et al. Blood. 2016.

Abstract

Intron retention (IR) is a form of alternative splicing that can impact mRNA levels through nonsense-mediated decay or by nuclear mRNA detention. A complex, dynamic IR pattern has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We globally assessed IR in primary maturing mammalian erythroid and megakaryocyte (MK) lineages as well as their common progenitor cells (MEPs). Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes with an overwhelming loss of IR in erythroid cells and MKs compared to MEPs. Moreover, complex IR patterns were seen throughout murine erythroid maturation. Similarly complex patterns were observed in human erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and MKs, and overlapping gene expression patterns, the MK IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. Importantly, our results suggest that IR serves to broadly regulate mRNA levels. These findings highlight the importance of this understudied form of alternative splicing in gene regulation and provide a useful resource for studies on gene expression in the MK and erythroid lineages.

Copyright © 2016 American Society of Hematology.

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Figures

Figure 1
Figure 1

Experimental strategy. (A) Purification strategy for samples used to generate these data sets. Baso, basophilic erythroblast; BFU-E, burst-forming unit-erythroid; BFU-MK, burst-forming unit-megakaryocyte; CFU-E, colony-forming unit-erythroid; CFU-MK, colony-forming unit-megakaryocyte; MEP, megakaryocyte-erythrocyte precursor; MK, megakaryocyte; Ortho, orthochromatic erythroblast; Poly, polychromatic erythroblast; Pro, proerythroblast. Purification strategies for MEPs, the MK lineage in bulk, the erythroid lineage in bulk, and individual erythroid stages are indicated. (B) Strategy to identify differentially retained introns between pairs of samples using IRFinder.

Figure 2
Figure 2

IR dynamics between MEPs and MKs or MEPs and erythroid cells. (A) IR changes between MEPs and MKs (upper panels) or erythroid cells (lower panels). Scatterplots depict IR ratios between pairs of samples for introns with significantly (Ben-Hoch, P < .05) different IR ratios. Bar graphs depict log2 IR ratio fold-changes. Number of introns with IR ratios increasing greater than or equal to twofold, decreasing greater than or equal to twofold, or changing less than twofold are indicated. Dashed bars indicate twofold changes. Empty regions along bar graph x-axes are introns with IR = 0 in 1 sample, their fold-changes cannot be plotted on a log-transformed scale. (B) Differential IR among both lineages. Panels indicate introns with greater than or equal to twofold IR ratio changes. (C) Histograms of introns per gene with greater than or equal to twofold IR ratio changes. Absolute frequencies are indicated above bars. (D) Introns with greater than or equal to twofold IR ratio fold-changes in both lineages. Scatterplot depicts log2 IR ratio fold-changes between MEPs and MKs (x-axis) and MEPs and erythroid cells (y-axis). Introns with IR ratio = 0 in one sample cannot have their fold-changes plotted on a log-transformed scale and are omitted.

Figure 3
Figure 3

Differential IR at multiple stages during murine erythroid maturation. (A) Bar graphs depicting IR ratio fold-changes between pairs of samples. Number of introns with IR ratios increasing greater than or equal to twofold, decreasing greater than or equal to twofold, or changing less than twofold are indicated. Dashed bars indicate twofold changes. (B) Number of introns and genes with IR ratios changing greater than or equal to twofold between proerythroblasts and each downstream stage. Total introns and genes among all pairwise comparisons is shown above. (C) Histograms of introns per gene with greater than or equal to twofold IR ratio changes. Absolute frequencies are indicated above bars. (D) Heat map of IR ratios across stages. Rows represent introns whose IR ratio changes greater than or equal to twofold between proerythroblasts and at least 1 downstream stage. Columns represent stages. Colors indicate Z-scores of IR ratios across each row. Rows were ordered using the Pearson distance metric and complete clustering method. Clusters were identified visually and using the dendrogram and are indicated to the right.

Figure 4
Figure 4

Experimental validation of differential IR. (A) Fluorescence-activated cell sorting profiles of unsorted and sorted murine erythroid marrow cells. Target antigens and fluorophores are indicated along axes. (B) Bar graph depicts quantitative reverse-transcription polymerase chain reaction of β-major (Hbb-b1) mRNA levels normalized to β-actin mRNA levels. (C) Genes and introns are indicated above, samples are indicated below, and the ratio of intron-exon signals (intron retained) to exon-exon signals (intron spliced out) are indicated to the left of each graph. Graphs depict mean and standard error of the mean of 4 to 6 biological replicates. Asterisks indicate significance (Wilcoxon rank-sum test, P < .05). Arrows indicate quantitative polymerase chain reaction primer locations.

Figure 5
Figure 5

Differential IR at multiple stages during human erythroid maturation. (A) Bar graphs depicting IR ratio fold-changes between pairs of samples. Number of introns with IR ratios increasing greater than or equal to twofold, decreasing greater than or equal to twofold, or changing less than twofold are indicated. Dashed bars indicate twofold changes. (B) Number of introns and genes with IR ratios changing greater than or equal to twofold between proerythroblasts and each downstream stage. Total introns and genes among all pairwise comparisons is shown above. (C) Histograms of introns per gene with greater than or equal to twofold IR ratio changes. Absolute frequencies are indicated above bars. (D) Heat map of IR ratios across stages. Rows represent introns whose IR ratio changes greater than or equal to twofold between proerythroblasts and at least 1 downstream stage. Columns represent stages. Colors indicate Z-scores of IR ratios across each row. Rows were ordered using the Pearson distance metric and complete clustering method. Clusters were identified visually and using the dendrogram and are indicated to the right. EB, early basophilic erythroblast; LB, late basophilic erythroblast.

Figure 6
Figure 6

An inverse correlation between gene expression changes and IR changes. Graphs depict log2 expression changes between pairs of samples for all genes with measured IR ratios (center panels), genes with IR ratios only increasing greater than or equal to twofold (left panels), and genes with IR ratios only decreasing greater than or equal to twofold (right panels). Pairwise comparisons of samples are indicated to the left. Median log2 expression changes are indicated within each graph.

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