We have systematically identified the targets of the Schizosaccharomyces pombe RNA-binding protein Meu5p, which is transiently induced during cellular differentiation. Meu5p-bound transcripts (>80) are expressed at low levels and have shorter half-lives in meu5 mutants, suggesting that Meu5p binding stabilizes its RNA targets.Most Meu5p targets are induced during differentiation by the activity of the Mei4p transcription factor. However, although most Mei4p targets display a sharp peak of expression, Meu5p targets are expressed for a longer period. In the absence of Meu5p, all Mei4p targets are expressed with similar kinetics (similar to non-Meu5p targets). Therefore, Meu5p determines the temporal profile of its targets.As the meu5 gene is itself a target of the transcription factor Mei4p, the RNA-binding protein Meu5p and their shared targets form a feed-forward loop (FFL), a network motif that is common in transcriptional networks.Our data highlight the importance of considering both transcriptional and posttranscriptional controls to understand dynamic changes in RNA levels, and provide insight into the structure of the regulatory networks that integrate transcription and RNA decay.
RNA levels are determined by the balance between RNA production (transcription) and degradation (decay or turnover). Therefore, cells can alter transcript levels by modulating either or both processes. Regulation of transcriptional initiation is one of the most common ways to regulate RNA levels. This function is frequently performed by transcription factors (TFs), which recognize specific sequence motifs on the promoters of their target genes and activate or repress their transcription. At the posttranscriptional level, RNA-binding proteins (RBPs) can bind to specific sequences on their target RNAs and regulate their rates of turnover.
RNA decay can be studied at the genome-wide level using microarrays or next-generation sequencing. The contribution of RNA turnover to transcript levels can be assessed by directly measuring decay rates. This is usually achieved by using microarrays to follow the decrease of RNA levels after inactivation of RNA polymerase II, or by in vivo labelling of newly synthesized RNA with modified nucleosides. These approaches can be applied to mutants in genes encoding RBPs, allowing the dissection of their specific functions in RNA turnover. Moreover, direct RBP targets can be identified by purifying RBP–RNA complexes, which are then analysed using microarrays (RIp-chip, for RBP Immunoprecipitation followed by analysis with DNA chips).
Many biological processes involve the establishment of complex programs of gene expression, in which the levels of hundreds of mRNAs are dynamically regulated. Although the genome-wide function of TFs in these processes has been studied extensively, much less is known about the contribution of RBPs, and especially about how the activity of TFs and RBPs is coordinated. Sexual differentiation of the fission yeast Schizosaccharomyces pombe culminates in meiosis and sporulation and is driven by an extensive gene expression program during which ∼40% of the genome (∼2000 genes) is regulated in complex temporal patterns. Transcriptional control is essential for the implementation of this program, and TFs responsible for the induction of most groups of upregulated genes have been identified. In particular, a transcription factor called Mei4p, which is itself transiently expressed during the meiotic divisions, induces the temporary expression of over 500 genes.
Here, we use genome-wide approaches to investigate the function of the Meu5p RBP, which is transiently induced by the Mei4p TF during the meiotic divisions. RIp-chip experiments identified >80 transcripts bound to Meu5p during meiosis, most of which were also targets of the Mei4p transcription factor. In meu5 mutants, Meu5p targets are expressed at low levels and have shorter half-lives, indicating that Meu5p stabilizes the transcripts it binds to. This stabilization has biological importance, as cells without meu5 are defective in spore formation.
Although the majority of Mei4p TF targets reach their peak in expression levels with similar kinetics, we noticed that the timing of their downregulation was heterogeneous. We could identify two discrete groups among Mei4p targets: a set of mRNAs with short (∼1 h) and sharp gene expression profiles (early decrease), and a group that displayed a broader expression pattern, with high levels of expression for 2–3 h (late decrease).
Most Meu5p RBP targets belonged to the late-decrease group, suggesting a simple model in which Meu5p might stabilize its targets, thus extending the duration of their expression. To test this idea, we followed gene expression in synchronized cultures of wild-type and meu5Δ meiotic cells. Although the expression of early decrease genes was not affected by the absence of meu5, late-decrease genes switched their profile to a pattern similar to that of early decrease genes. As transcription of meu5 is under the control of Mei4p, the TF Mei4p, the RBP Meu5p, and their common targets form a so-called feed-forward loop, in which a protein regulates a target both directly and indirectly through a second protein. This arrangement is common in transcriptional and protein phosphorylation networks.
Our results serve as a paradigm of how the coordination of the action of TFs and RBPs determines how RNA levels are dynamically regulated.
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 ∼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.