Global coordination of transcriptional control and mRNA decay during cellular differentiation

Abstract

The function of transcription in dynamic gene expression programs has been extensively studied, but little is known about how it is integrated with RNA turnover at the genome-wide level. We investigated these questions using the meiotic gene expression program of Schizosaccharomyces pombe. We identified over 80 transcripts that co-purify with the meiotic-specific Meu5p RNA-binding protein. Their levels and half-lives were reduced in meu5 mutants, demonstrating that Meu5p stabilizes its targets. Most Meu5p-bound RNAs were also targets of the Mei4p transcription factor, which induces the transient expression of approximately 500 meiotic genes. Although many Mei4p targets showed sharp expression peaks, Meu5p targets had broad expression profiles. In the absence of meu5, all Mei4p targets were expressed with similar kinetics, indicating that Meu5p alters the global features of the gene expression program. As Mei4p activates meu5 transcription, Mei4p, Meu5p and their common targets form a feed-forward loop, a motif common in transcriptional networks but not studied in the context of mRNA decay. Our data provide insight into the topology of regulatory networks integrating transcriptional and posttranscriptional controls.

Figure 1

Meu5p has a function in sporulation and regulates the expression of a subset of meiotic middle genes. (A) Wild-type or meu5Δ h90 cells were incubated for 24 h in the absence of a nitrogen source to induce sexual differentiation and visualized using phase contrast microscopy. (B) Comparison of expression levels between wild-type and meu5Δ in pat1-synchronized meiotic diploid cells. Middle genes are shown in red and other genes in grey. Genes outside the dashed lines differ by more than two-fold in expression levels. (C) Overlap between genes downregulated in meu5Δ diploids (in pat1-synchronized meiosis) and middle genes. The numbers in brackets show the overlap between the two lists expected by chance given the sizes of the gene sets considered and the total number of genes. The P-value of the overlap is shown under the Venn diagram.

Figure 2

Meu5p associates with transcripts encoded by middle genes and regulates their expression. (A) Meu5p targets are expressed at low levels in meu5Δ cells. Comparison of expression levels between wild-type and meu5Δ pat1-synchronized meiotic cells (data as in Figure 1B). Meu5p-associated transcripts identified by RIp-chip are shown in red and other genes are shown in grey. The position of the meu5 transcript is indicated with an arrow. (B) Overlap between Meu5p-associated transcripts and middle genes. (C) Overlap between Meu5p-associated transcripts and genes downregulated in meu5Δ cells (in pat1-synchronized meiosis). Labelling of the Venn diagrams is as in Figure 1C.

Figure 3

Meu5p stabilizes its targets. (A) Comparison of the fraction of newly synthesized RNA (4sU-labelled) between wild-type and meu5Δ pat1-synchronized meiotic cells. Meu5p-associated transcripts are shown in red and other mRNAs are shown in grey. Genes outside the dashed lines differ by more than two-fold in labelling levels. Note that a higher fraction corresponds to a shorter half-life. (B) Overlap between genes destabilized in meu5Δ cells, genes downregulated in meu5Δ diploid cells (in pat1-synchronized meiosis) and Meu5p-associated transcripts. The numbers outside the Venn diagrams show the P-values of the pairwise comparisons.

Figure 4

Meu5p targets have distinct meiotic expression profiles. (A) Meiotic gene expression profiles of Meu5p targets. Vegetatively growing cells (V) are synchronized in G1 by nitrogen removal and enter meiosis by inactivation of pat1 at time 0 (expression data are from Mata et al, 2002). The expression profiles of mei4 (orange) and meu5 (green) are also shown. Note that neither meu5 nor mei4 transcripts are bound by Meu5p. (B, C) Classification of middle genes according to the kinetics of their downregulation (expression data are from Mata et al, 2002). ‘Early decrease’ genes are shown in red and ‘late-decrease’ are shown in blue. Average expression profiles for each group are shown in (B) and profiles for all genes are shown in (C). (D) Overlap between Meu5p-associated transcripts and ‘early-decrease’ genes (left) or ‘late decrease’ (right). Labelling of the Venn diagrams is as in Figure 1C.

Figure 5

Meu5p regulates the dynamics of expression of its targets. (A) Average expression profiles of ‘early-decrease’ (left) or ‘late-decrease’ (right) genes in pat1-induced meiotic time courses. Labelling of the graphs is as in Figure 4A, except that expression ratios were normalized to the levels at 3 h after the induction of meiosis in the corresponding experiment. Data from wild-type meiosis are shown in purple and from meu5Δ are shown in green. (B) Diagram summarizing the regulation of the expression of middle genes. Proteins are shown as ovals and transcripts are shown as curvy lines. All middle genes are induced transcriptionally by Mei4p. Meu5p stabilizes its targets allowing them to be expressed for longer, whereas middle genes not bound by Meu5p are induced more transiently. Mei4p induces the expression of meu5, and thus Mei4p, Meu5p and their common targets form a feed-forward network motif.