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Regulation of mRNA translation during mitosis.

Tanenbaum ME, Stern-Ginossar N, Weissman JS, Vale RD - Elife (2015)

Bottom Line: Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated.One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis.We show that full APC activation requires translational repression of Emi1 in addition to its degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

No MeSH data available.


Related in: MedlinePlus

Excessive PP1γ and PP2aβ activity perturbs chromosome segregation.(A–D) RPE-1 cells expressing either H2B-GFP alone or together with mCherry and PP1γ (A, C) or mCherry and PP2aβ (B, D) were analyzed by time-lapse microscopy. Simultaneous expression of mCherry and untagged PP1γ or PP2aβ was accomplished by inserting a P2A ribosome skipping sequence in between mCherry and the phosphatase sequence. (A) Stills from representative videos of control cell (upper panel) or PP1γ overexpressing cell (lower panel). Time is shown in min. (B) Stills from representative video of control cell (upper panel) or PP2aβ expressing cells (lower two panels). The top PP2aβ expressing cell shows a prometaphase arrest with misaligned chromosomes, while the bottom cell shows a cell with a prometaphase delay and subsequent cytokinesis without chromosome segregation, known as a ‘cut’ phenotype. Time is shown in min. (C) The fraction of cells in which one or more chromosomes mis-segregated was determined for control cells and cells expressing PP1γ. (D) shows the average time from NEB to anaphase for control cells and cells expressing PP2aβ. All graphs are the mean and SD of 3 independent experiments with 20–40 cells analyzed per experiment. Scale bars, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.07957.007
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fig2s3: Excessive PP1γ and PP2aβ activity perturbs chromosome segregation.(A–D) RPE-1 cells expressing either H2B-GFP alone or together with mCherry and PP1γ (A, C) or mCherry and PP2aβ (B, D) were analyzed by time-lapse microscopy. Simultaneous expression of mCherry and untagged PP1γ or PP2aβ was accomplished by inserting a P2A ribosome skipping sequence in between mCherry and the phosphatase sequence. (A) Stills from representative videos of control cell (upper panel) or PP1γ overexpressing cell (lower panel). Time is shown in min. (B) Stills from representative video of control cell (upper panel) or PP2aβ expressing cells (lower two panels). The top PP2aβ expressing cell shows a prometaphase arrest with misaligned chromosomes, while the bottom cell shows a cell with a prometaphase delay and subsequent cytokinesis without chromosome segregation, known as a ‘cut’ phenotype. Time is shown in min. (C) The fraction of cells in which one or more chromosomes mis-segregated was determined for control cells and cells expressing PP1γ. (D) shows the average time from NEB to anaphase for control cells and cells expressing PP2aβ. All graphs are the mean and SD of 3 independent experiments with 20–40 cells analyzed per experiment. Scale bars, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.07957.007

Mentions: Next, we examined whether there were particularly types of genes that were predominantly regulated by translational vs transcriptional control, so we performed gene ontology enrichment analysis using the functional annotation tool DAVID (Huang da et al., 2009). Many genes that exhibited variations in mRNA levels during the cell cycle are involved in cell division (p-values < 10−9, see ‘Materials and methods’). In contrast, the translationally regulated genes were functionally very different from the transcriptionally regulated genes and included many signaling molecules, transcription factors, and transmembrane proteins (significantly enriched with p-values < 10−8) (see Figure 2—figure supplement 2). Manual curation of the mRNAs that showed gene-specific translational repression during mitosis revealed regulation of several components of the same pathway. For example, multiple components of the PI3 kinase pathway were translationally repressed during mitosis (Figure 2—figure supplement 2A). We also found mitosis-specific translational downregulation of the mitotic phosphatases PP1γ and PP2aβ (Figure 2—figure supplement 2B). During mitosis, these phosphatases are strongly inhibited through phosphorylation and binding to inhibitory proteins (Wurzenberger and Gerlich, 2011), suggesting that mitotic translational repression may represent an additional back-up mechanism to inhibit protein function (see Discussion). Consistent with this notion, we found that overexpression of either PP1γ or PP2aβ phosphatase strongly disrupted normal cell division (Figure 2—figure supplement 3A–C). We also found a strong translational repression of two key regulators of centriole duplication, Plk4 and CP110 (Figure 2—figure supplement 2C) (Chen et al., 2002; Habedanck et al., 2005). Finally, the majority of histones showed a strong reduction in protein synthesis in M compared with G2. Newly synthesized histones may not incorporate readily into highly condensed mitotic chromosomes, which perhaps could explain why their translation is reduced during mitosis, although we cannot completely rule out a small contamination of S-phase cells in the G2 sample which might give rise to an apparent high level of translation of histone mRNAs in G2. We also found a few mRNAs that were translationally increased in mitosis as compared to both G1 and G2, although most were below our threshold of threefold change, indicating the changes were subtle. Included in this list are genes involved in cytoskeleton function and DNA replication initiation (Figure 2—figure supplement 2E,F), the latter of which may reflect the ability of cells to license DNA replication at the end of mitosis (Clijsters et al., 2013). Taken together, these results show that transcriptional and gene-specific translational control dominate at different stages of the cell cycle (transcription at the G1-to-G2 transition and translational regulation dominating at the more rapid G2-to-M transition) and regulate a distinct set of genes.


Regulation of mRNA translation during mitosis.

Tanenbaum ME, Stern-Ginossar N, Weissman JS, Vale RD - Elife (2015)

Excessive PP1γ and PP2aβ activity perturbs chromosome segregation.(A–D) RPE-1 cells expressing either H2B-GFP alone or together with mCherry and PP1γ (A, C) or mCherry and PP2aβ (B, D) were analyzed by time-lapse microscopy. Simultaneous expression of mCherry and untagged PP1γ or PP2aβ was accomplished by inserting a P2A ribosome skipping sequence in between mCherry and the phosphatase sequence. (A) Stills from representative videos of control cell (upper panel) or PP1γ overexpressing cell (lower panel). Time is shown in min. (B) Stills from representative video of control cell (upper panel) or PP2aβ expressing cells (lower two panels). The top PP2aβ expressing cell shows a prometaphase arrest with misaligned chromosomes, while the bottom cell shows a cell with a prometaphase delay and subsequent cytokinesis without chromosome segregation, known as a ‘cut’ phenotype. Time is shown in min. (C) The fraction of cells in which one or more chromosomes mis-segregated was determined for control cells and cells expressing PP1γ. (D) shows the average time from NEB to anaphase for control cells and cells expressing PP2aβ. All graphs are the mean and SD of 3 independent experiments with 20–40 cells analyzed per experiment. Scale bars, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.07957.007
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fig2s3: Excessive PP1γ and PP2aβ activity perturbs chromosome segregation.(A–D) RPE-1 cells expressing either H2B-GFP alone or together with mCherry and PP1γ (A, C) or mCherry and PP2aβ (B, D) were analyzed by time-lapse microscopy. Simultaneous expression of mCherry and untagged PP1γ or PP2aβ was accomplished by inserting a P2A ribosome skipping sequence in between mCherry and the phosphatase sequence. (A) Stills from representative videos of control cell (upper panel) or PP1γ overexpressing cell (lower panel). Time is shown in min. (B) Stills from representative video of control cell (upper panel) or PP2aβ expressing cells (lower two panels). The top PP2aβ expressing cell shows a prometaphase arrest with misaligned chromosomes, while the bottom cell shows a cell with a prometaphase delay and subsequent cytokinesis without chromosome segregation, known as a ‘cut’ phenotype. Time is shown in min. (C) The fraction of cells in which one or more chromosomes mis-segregated was determined for control cells and cells expressing PP1γ. (D) shows the average time from NEB to anaphase for control cells and cells expressing PP2aβ. All graphs are the mean and SD of 3 independent experiments with 20–40 cells analyzed per experiment. Scale bars, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.07957.007
Mentions: Next, we examined whether there were particularly types of genes that were predominantly regulated by translational vs transcriptional control, so we performed gene ontology enrichment analysis using the functional annotation tool DAVID (Huang da et al., 2009). Many genes that exhibited variations in mRNA levels during the cell cycle are involved in cell division (p-values < 10−9, see ‘Materials and methods’). In contrast, the translationally regulated genes were functionally very different from the transcriptionally regulated genes and included many signaling molecules, transcription factors, and transmembrane proteins (significantly enriched with p-values < 10−8) (see Figure 2—figure supplement 2). Manual curation of the mRNAs that showed gene-specific translational repression during mitosis revealed regulation of several components of the same pathway. For example, multiple components of the PI3 kinase pathway were translationally repressed during mitosis (Figure 2—figure supplement 2A). We also found mitosis-specific translational downregulation of the mitotic phosphatases PP1γ and PP2aβ (Figure 2—figure supplement 2B). During mitosis, these phosphatases are strongly inhibited through phosphorylation and binding to inhibitory proteins (Wurzenberger and Gerlich, 2011), suggesting that mitotic translational repression may represent an additional back-up mechanism to inhibit protein function (see Discussion). Consistent with this notion, we found that overexpression of either PP1γ or PP2aβ phosphatase strongly disrupted normal cell division (Figure 2—figure supplement 3A–C). We also found a strong translational repression of two key regulators of centriole duplication, Plk4 and CP110 (Figure 2—figure supplement 2C) (Chen et al., 2002; Habedanck et al., 2005). Finally, the majority of histones showed a strong reduction in protein synthesis in M compared with G2. Newly synthesized histones may not incorporate readily into highly condensed mitotic chromosomes, which perhaps could explain why their translation is reduced during mitosis, although we cannot completely rule out a small contamination of S-phase cells in the G2 sample which might give rise to an apparent high level of translation of histone mRNAs in G2. We also found a few mRNAs that were translationally increased in mitosis as compared to both G1 and G2, although most were below our threshold of threefold change, indicating the changes were subtle. Included in this list are genes involved in cytoskeleton function and DNA replication initiation (Figure 2—figure supplement 2E,F), the latter of which may reflect the ability of cells to license DNA replication at the end of mitosis (Clijsters et al., 2013). Taken together, these results show that transcriptional and gene-specific translational control dominate at different stages of the cell cycle (transcription at the G1-to-G2 transition and translational regulation dominating at the more rapid G2-to-M transition) and regulate a distinct set of genes.

Bottom Line: Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated.One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis.We show that full APC activation requires translational repression of Emi1 in addition to its degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

No MeSH data available.


Related in: MedlinePlus