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1.  Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana 
A protein interactome focused towards cell proliferation was mapped comprising 857 interactions among 393 proteins, leading to many new insights in plant cell cycle regulation.A comprehensive view on heterodimeric cyclin-dependent kinase (CDK)/cyclin complexes in plants is obtained, in relation with their regulators.Over 100 new candidate cell cycle proteins were predicted.
The basic underlying mechanisms that govern the cell cycle are conserved among all eukaryotes. Peculiar for plants, however, is that their genome contains a collection of cell cycle regulatory genes that is intriguingly large (Vandepoele et al, 2002; Menges et al, 2005) compared to other eukaryotes. Arabidopsis thaliana (Arabidopsis) encodes 71 genes in five regulatory classes versus only 15 in yeast and 23 in human.
Despite the discovery of numerous cell cycle genes, little is known about the protein complex machinery that steers plant cell division. Therefore, we applied tandem affinity purification (TAP) approach coupled with mass spectrometry (MS) on Arabidopsis cell suspension cultures to isolate and analyze protein complexes involved in the cell cycle. This approach allowed us to successfully map a first draft of the basic cell cycle complex machinery of Arabidopsis, providing many new insights into plant cell division.
To map the interactome, we relied on a streamlined platform comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and matrix-assisted laser desorption ionization (MALDI) tandem-MS for the identification of purified proteins (Van Leene et al, 2007, 2008Van Leene et al, 2007, 2008). Complexes for 102 cell cycle proteins were analyzed using this approach, leading to a non-redundant data set of 857 interactions among 393 proteins (Figure 1A). Two subspaces were identified in this data set, domain I1, containing interactions confirmed in at least two independent experimental repeats or in the reciprocal purification experiment, and domain I2 consisting of uniquely observed interactions.
Several observations underlined the quality of both domains. All tested reverse purifications found the original interaction, and 150 known or predicted interactions were confirmed, meaning that also a huge stack of new interactions was revealed. An in-depth computational analysis revealed enrichment for many cell cycle-related features among the proteins of the network (Figure 1B), and many protein pairs were coregulated at the transcriptional level (Figure 1C). Through integration of known cell cycle-related features, more than 100 new candidate cell cycle proteins were predicted (Figure 1D). Besides common qualities of both interactome domains, their real significance appeared through mutual differences exposing two subspaces in the cell cycle interactome: a central regulatory network of stable complexes that are repeatedly isolated and represent core regulatory units, and a peripheral network comprising transient interactions identified less frequently, which are involved in other aspects of the process, such as crosstalk between core complexes or connections with other pathways. To evaluate the biological relevance of the cell cycle interactome in plants, we validated interactions from both domains by a transient split-luciferase assay in Arabidopsis plants (Marion et al, 2008), further sustaining the hypothesis-generating power of the data set to understand plant growth.
With respect to insights into the cell cycle physiology, the interactome was subdivided according to the functional classes of the baits and core protein complexes were extracted, covering cyclin-dependent kinase (CDK)/cyclin core complexes together with their positive and negative regulation networks, DNA replication complexes, the anaphase-promoting complex, and spindle checkpoint complexes. The data imply that mitotic A- and B-type cyclins exclusively form heterodimeric complexes with the plant-specific B-type CDKs and not with CDKA;1, whereas D-type cyclins seem to associate with CDKA;1. Besides the extraction of complexes previously shown in other organisms, our data also suggested many new functional links; for example, the link coupling cell division with the regulation of transcript splicing. The association of negative regulators of CDK/cyclin complexes with transcription factors suggests that their role in reallocation is not solely targeted to CDK/cyclin complexes. New members of the Siamese-related inhibitory proteins were identified, and for the first time potential inhibitors of plant-specific mitotic B-type CDKs have been found in plants. New evidence that the E2F–DP–RBR network is not only active at G1-to-S, but also at the G2-to-M transition is provided and many complexes involved in DNA replication or repair were isolated. For the first time, a plant APC has been isolated biochemically, identifying three potential new plant-specific APC interactors, and finally, complexes involved in the spindle checkpoint were isolated mapping many new but specific interactions.
Finally, to get a general view on the complex machinery, modules of interacting cyclins and core cell cycle regulators were ranked along the cell cycle phases according to the transcript expression peak of the cyclins, showing an assorted set of CDK–cyclin complexes with high regulatory differentiation (Figure 4). Even within the same subfamily (e.g. cyclin A3, B1, B2, D3, and D4), cyclins differ not only in their functional time frame but also in the type and number of CDKs, inhibitors, and scaffolding proteins they bind, further indicating their functional diversification. According to our interaction data, at least 92 different variants of CDK–cyclin complexes are found in Arabidopsis.
In conclusion, these results reflect how several rounds of gene duplication (Sterck et al, 2007) led to the evolution of a large set of cyclin paralogs and a myriad of regulators, resulting in a significant jump in the complexity of the cell cycle machinery that could accommodate unique plant-specific features such as an indeterminate mode of postembryonic development. Through their extensive regulation and connection with a myriad of up- and downstream pathways, the core cell cycle complexes might offer the plant a flexible toolkit to fine-tune cell proliferation in response to an ever-changing environment.
Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)–cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK–cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.
doi:10.1038/msb.2010.53
PMCID: PMC2950081  PMID: 20706207
Arabidopsis thaliana; cell cycle; interactome; protein complex; protein interactions
2.  Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex 
eLife  null;5:e20934.
Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.
DOI: http://dx.doi.org/10.7554/eLife.20934.001
eLife digest
Cells use the genetic information stored within genes to build proteins, which are largely responsible for performing the molecular tasks essential for life. The ribosome is the molecular machine that translates the information within genes to assemble proteins in all cells, from bacteria to humans. To make a protein, the corresponding gene is first copied to make molecules of messenger ribonucleic acid (or mRNA for short). Then the ribosome binds to the mRNA in a process called translation initiation.
Cells tightly regulate translation initiation so that they can decide which proteins to make, according to their needs and in response to changes in the environment. In fact, regulation of translation initiation is often disrupted during viral infections, cancer and other human diseases. A set of proteins called translation initiation factors drive translation initiation; the largest and least understood of these is called eIF3.
Cells are unable to load the mRNA onto the ribosome without eIF3, which has two “arms” that sit near where the mRNA enters and exits the ribosome. Aitken et al. used mutant forms of eIF3 from genetically modified yeast to investigate how the arms of the protein work, and if they help the ribosome hold onto the mRNA.
These experiments show that the two arms of eIF3 have unique roles. One arm sits near where mRNA exits the ribosome and is important for holding onto the mRNA. The other arm – which is near where mRNA enters the ribosome – helps hold the ribosome and other components of the translation machinery together. This arm may also help to open and close the channel through which messenger RNA enters the ribosome. The next challenges are to find out the precise role this arm plays in translation – in particular, how it helps to open and close the channel in the ribosome, and whether this helps the ribosome load the messenger RNA or even move along it.
DOI: http://dx.doi.org/10.7554/eLife.20934.002
doi:10.7554/eLife.20934
PMCID: PMC5153249  PMID: 27782884
eIF3; translation; initiation; ribosome; yeast; mRNA recruitment; S. cerevisiae
3.  Commitment Point during G0→G1 That Controls Entry into the Cell Cycle 
Molecular and Cellular Biology  2003;23(7):2351-2361.
Initiation of T-lymphocyte-mediated immune responses involves two cellular processes: entry into the cell cycle (G0→G1) for clonal proliferation and coordinated changes in surface and secreted molecules that mediate effector functions. However, a point during G0→G1 beyond which T cells are committed to enter the cell cycle has not been defined. We define here a G0→G1 commitment point that occurs 3 to 5 h after CD3 and CD28 stimulation of human CD4 or CD8 T cells. Transition through this point requires cdk6/4-cyclin D, since inhibition with TAT-p16INK4A during the first 3 to 5 h prevents cell cycle entry and maintains both naive and memory T cells in G0. Transition through the G0→G1 commitment point is also necessary for T cells to increase in size, i.e., to enter the cellular growth cycle. However, transition through this point is not required for the induction of effector functions. These can be initiated while cells are maintained in G0 with TAT-p16INK4A. We have termed this quiescent, activated state G0(A). Our data provide proof of the principle that entry of T cells into the cell cycle and cellular growth cycles are coupled at the G0→G1 commitment point but that these processes can be uncoupled from the early expression of molecules of effector functions.
doi:10.1128/MCB.23.7.2351-2361.2003
PMCID: PMC150729  PMID: 12640120
4.  Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy 
PLoS Medicine  2006;3(12):e528.
Background
Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum.
Methods and Findings
We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.
Conclusions
Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.
Erythrocyte G protein signaling is needed for intracellular malarial parasite proliferation and thus may present a novel antimalarial target.
Editors' Summary
Background.
New drugs for treatment of malaria are urgently needed, because the malaria parasite has evolved resistance against virtually all types of commonly used drugs. When a person is bitten by a malaria-infected mosquito, the parasite first infects the person's liver cells before going on to infect red blood cells, where the parasites multiply and develop into a parasite stage called a schizont. The red blood cells then burst and release more schizonts into the bloodstream; it is this “blood stage” of infection in humans that causes the symptoms of disease. Therefore efforts to develop new drugs against malaria often focus on this “blood stage” of infection. One strategy for developing new drugs is termed the “host-targeted” approach. This means that rather than trying to block processes occurring within the parasite itself, a drug can be developed which blocks processes within the person's red blood cells, and which would otherwise be needed for the parasite to complete its life cycle. It will be difficult for malaria parasites to evolve resistance to such a drug, because changes in a person's red blood cells occur much more slowly than in the parasites themselves.
Why Was This Study Done?
This research group has been studying a set of molecular processes within human red blood cells which seemed to be required for entry of malaria parasites into the cells. They wanted to get a better understanding of those processes and, specifically, to find out whether it would be possible to use particular molecules to block those processes, and by doing so to prevent malaria parasites from entering and multiplying within red blood cells. In particular, when the malaria parasites invade the red blood cell, they form membranes around the red blood cell, containing lipids and proteins “hijacked” from the red blood cell membrane. These researchers already knew that two particular proteins were hijacked in this way; the β2-adrenergic receptor (β2-AR) and heterotrimeric G protein (Gs). These two proteins act together to pass messages across the surface of the membrane to inside the cell. Small molecules could be used to block signaling through β2-AR and Gs, and therefore potentially to provide a new way of preventing malaria parasites from entering red blood cells and multiplying within them.
What Did the Researchers Do and Find?
Firstly, the researchers made red blood cell “ghosts” in which to study these molecular processes. This meant that they took fresh red blood cells from healthy human volunteers, burst them to remove half the contents and loaded them with markers and other cargoes before resealing the membranes of the cell. These resealed markers and cargoes allowed them to see what was happening inside the cells. Malaria parasites were able to invade these ghosts normally and multiply within them. When the researchers introduced a specific peptide (a molecule consisting of a short series of amino acids), they found that it blocked Gs signaling within the ghosts. This peptide also prevented malaria parasites from developing inside the ghosts. Therefore, they concluded that Gs signaling inside the red blood cell was important for the parasite life cycle. The researchers then examined a drug called propranolol which is already known to act on Gs signaling and which is commonly prescribed for high blood pressure. This drug also blocked development of malaria parasites inside the ghosts when used at a particular concentration. Finally, the researchers studied the effect of giving propranolol, along with other antimalarial drugs, to human malaria parasites in a culture dish and to mice injected with a malaria parasite that infects rodents. In these experiments, adding propranolol reduced the amount of other “parasite-targeted” drugs that were needed to effectively treat malarial infection in tissue culture and in mice.
What Do These Findings Mean?
Showing that the Gs signaling pathway is important for the malaria parasite's life cycle opens up new possibilities for drug development. Specifically, propranolol (which is already approved for treatment of high blood pressure and other conditions) might itself provide a new candidate therapy, either alone or in combination with existing drugs. These combinations would first, however, need to be tested in human clinical trials, perhaps by seeing whether they have antimalarial activity in people who have not responded to existing antimalarial drugs. Since it acts to lower blood pressure, which can already be low in some people with malaria, there are some concerns that propranolol might not be a suitable drug candidate for use, especially with existing antimalarial drugs that also reduce blood pressure. However, other molecules which block Gs signaling could be tested for activity against malaria should propranolol prove not to be an ideal drug candidate.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030528.
The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programs for malaria control
Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria
Wikipedia entries for drug discovery and drug development (Wikipedia is an internet encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030528
PMCID: PMC1716186  PMID: 17194200
5.  Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast 
PLoS Genetics  2016;12(6):e1006088.
Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF), which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state.
Author Summary
In spite of the many differences between yeast and humans, the basic strategies that regulate the cell division cycle are fundamentally conserved. In this study, we extend these parallels to include a common strategy by which cells transition from proliferation to quiescence. The decision to divide is made in the G1 phase of the cell cycle. During G1, the genes that drive DNA replication are repressed by the E2F/RB complex. When a signal to divide is received, RB is removed and the complex is activated. When cells commit to a long term, but reversible G1 arrest, or quiescence, they express a novel E2F/RB-like complex, which promotes and maintains a stable repressive state. Yeast cells contain a functional analog of E2F/RB, called SBF/Whi5, which is activated by a similar mechanism in proliferating yeast cells. In this study, we identify two novel components of the SBF/Whi5 complex whose activity is specific to the transition to quiescence. These factors, Msa1 and Msa2, repress SBF targets and are required for the long term, but reversible G1 arrest that is critical for achieving a quiescent state.
doi:10.1371/journal.pgen.1006088
PMCID: PMC4894574  PMID: 27272642
6.  A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts 
Genome Biology  2012;13(12):R121.
Background
Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.
Results
Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.
Conclusions
microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.
doi:10.1186/gb-2012-13-12-r121
PMCID: PMC3924601  PMID: 23259597
MicroRNA; Quiescence; Cell cycle; Proliferation; Extracellular matrix; Fibroblast; Microarray; miR-29
7.  The two eIF4A helicases in Trypanosoma brucei are functionally distinct 
Nucleic Acids Research  2006;34(9):2495-2507.
Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5′ capped mini-exon which is linked to the 3′ cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 × 105 molecules/cell) and depletion to ∼10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (∼1–2 × 104 molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.
doi:10.1093/nar/gkl290
PMCID: PMC1459412  PMID: 16687655
8.  Quiescent Fibroblasts Exhibit High Metabolic Activity 
PLoS Biology  2010;8(10):e1000514.
Metabolomics technology reveals that fibroblast that have exited the proliferative cell cycle nevertheless utilize glucose throughout central carbon metabolism and rely on the pentose phosphate pathway for viability.
Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.
Author Summary
Many cells in the human body are in a reversible state of quiescence, where they have exited the cell cycle but retain the capacity to re-enter it and divide again. Previous experiments in lymphocytes had suggested that quiescent cells reduce their glucose uptake and metabolic rate. In our studies, we have investigated the metabolism of fibroblasts, cells found in connective tissue and skin. Using “metabolomics” to monitor flux through metabolic pathways, we discovered that fibroblasts remain highly metabolically active even though they are not dividing. They degrade and resynthesize protein and fatty acid, and secrete large amounts of protein into the extracellular environment. Despite our expectation that quiescent cells would not have a high demand for nucleotide biosynthesis, we found that they do divert glucose to the pentose phosphate pathway, presumably to generate NADPH. The NADPH created may help the quiescent fibroblasts to detoxify free radicals or to synthesize fatty acids. Experiments in which we inhibited the pentose phosphate pathway resulted in increased apoptosis in quiescent cells, suggesting a possible strategy for selectively killing nondividing cells.
doi:10.1371/journal.pbio.1000514
PMCID: PMC2958657  PMID: 21049082
9.  Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension 
Molecular Biology of the Cell  1998;9(11):3179-3193.
The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.
PMCID: PMC25607  PMID: 9802905
10.  An extensive program of periodic alternative splicing linked to cell cycle progression 
eLife  null;5:e10288.
Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control.
DOI: http://dx.doi.org/10.7554/eLife.10288.001
eLife digest
Mitosis is a key step in the normal life cycle of a cell, during which one cell divides into two new cells. As a cell progresses through the cell cycle, it must carefully regulate its gene activity to switch particular genes on or off at specific moments. When a gene is activated its sequence is first copied into a temporary molecule called a transcript. These transcripts are then edited to form templates to build proteins. One way that a transcript can be edited is via a process called alternative splicing, in which different pieces of the transcript are cut and pasted together to form different versions of the final template. This allows different instructions to be obtained from a single gene, introducing an added layer of biological complexity. However, the role of alternative splicing in the timing of key events of the cell life cycle is not well understood.
Dominguez et al. have now looked for the genes that undergo alternative splicing during the cell cycle. The sequences of gene transcripts produced within human cells were collected while the cells went through two rounds of division. This approach revealed that around 1,300 genes are spliced in different ways at different stages of each cell cycle. Many of these genes were known to play roles in controlling the cell’s life cycle, but few of the genes showed large changes in the amount of total transcript that is generated over time.
Dominguez et al. also showed that an enzyme called CLK1 influences about half of the 1,300 periodically spliced genes during the cell cycle. The production of CLK1 is itself carefully controlled throughout the cell cycle, and the enzyme’s activity prevents its own overproduction. Further experiments showed that blocking CLK1’s activity while a cell is replicating its DNA halts the cell cycle, but blocking this enzyme’s activity after the cell had replicated its DNA did not. Given this pivotal role in the cell cycle, Dominguez et al. also examined the role of CLK1 in cancer cells and found that high levels of CLK1 in tumours were linked to lower survival rates. These findings indicate that CLK1 warrants further investigation, particularly in relation to its role in cancer.
DOI: http://dx.doi.org/10.7554/eLife.10288.002
doi:10.7554/eLife.10288
PMCID: PMC4884079  PMID: 27015110
alternative splicing; cancer genomic; cell cycle; Human
11.  Origin of bistability underlying mammalian cell cycle entry 
Mammalian cell cycle entry is controlled at the restriction point by a bistable and resettable switch, which is shown to emerge from a minimal gene circuit containing a mutual-inhibition feedback loop between Rb and E2F modules, coupled with a feed-forward loop between Myc and E2F modules.
A minimal gene circuit is identified underlying bistable cell cycle entry in mammalian cells by analyzing all possible link combinations in a simplified Rb–E2F signaling network.This minimal gene circuit contains a mutual-inhibition feedback loop between Rb and E2F modules, coupled with a feed-forward loop between Myc and E2F modules, which forms an AND-gate control of the E2F activation.Experimental disruption of this minimal gene circuit abolishes maintenance of the activated E2F state, supporting its importance for the bistability of Rb–E2F system.This minimal gene circuit suggests basic design principles for the robust control of the bistable cell cycle entry at the R-point.
The Rb–E2F pathway plays a critical role in controlling cell cycle entry and progression in mammalian cells. Deregulation of the RB–E2F pathway is implicated in most, if not all, human cancers (Nevins, 2001; Weinberg, 2007). Recently, we have demonstrated that the Rb–E2F pathway controls cell proliferation at the restriction point (R-point) by functioning as a bistable switch. This bistable switch converts graded and transient growth signals into an all-or-none activation of E2F activity. Once switched ON by growth stimulation, the E2F activity remains ON, even when the growth stimulation is diminished (Yao et al, 2008). Interestingly, the Rb–E2F bistable switch is intrinsically resettable: the steady-state E2F level is at the monostable OFF state at low-serum conditions (Yao et al, 2008). This resettability can provide a tight control over cell growth responses by limiting spontaneous cell cycle entry. Meanwhile, by creating a wide hysteresis loop and correspondingly long time delay, the bistability of the Rb–E2F switch can drive temporally irreversible R-point transition and subsequent cell cycle progression.
The resettable bistability of the Rb–E2F switch provides a mechanistic explanation of the R-point control between quiescence and cell proliferation. However, the design features that underlie this switching property have not been elucidated. Defining these features can provide insights into the essential control mechanism underlying mammalian cell cycle entry. Such a control mechanism could be conserved across various cell types while disrupted in most, if not all, cancer cells.
To this end, we constructed and analyzed a library of mathematical models that encompass all possible circuit designs derived from a simplified Rb–E2F network (Figure 1). We identified a minimal gene circuit that is uniquely robust in generating resettable bistability. This minimal circuit consists of a mutual-inhibition feedback loop between the Rb (RP) and E2F (EE) modules (Figure 1, links 5, 6) and a feed-forward loop between the Myc (MD) and E2F (EE) modules (Figure 1, links 7, 3, 6). These two regulatory motifs form an AND-gate control of E2F activation (system output). Our modeling analysis suggested that the mutual-inhibition feedback loop between the Rb and E2F modules is critical for generating a robust bistable switch. Meanwhile, the feed-forward loop between the Myc and E2F modules and the AND-gate control are critical for the resettability. Underscoring the importance of this model-predicted minimal circuit, targeted disruption of this circuit abolishes maintenance of the activated E2F state, supporting its necessity for the bistability of the Rb–E2F system (Figure 3).
The unique topology of the minimal circuit, by combining a mutual-inhibition feedback loop and a feed-forward loop into an AND-gate control of system output, also contributes to its structural flexibility in creating resettable bistability. This structural flexibility is manifest in the ability of the minimal circuit to often maintain resettable bistability, despite alterations in its network topology. This property could facilitate the system evolvability. Altogether, our study suggested a minimal gene circuit underlying the origin of the resettable bistability in the Rb–E2F network, which controls normal cell cycle entry of mammalian cells. Consistent with this notion, this minimal gene circuit appears targeted and disrupted by frequent mutations in human cancers.
Precise control of cell proliferation is fundamental to tissue homeostasis and differentiation. Mammalian cells commit to proliferation at the restriction point (R-point). It has long been recognized that the R-point is tightly regulated by the Rb–E2F signaling pathway. Our recent work has further demonstrated that this regulation is mediated by a bistable switch mechanism. Nevertheless, the essential regulatory features in the Rb–E2F pathway that create this switching property have not been defined. Here we analyzed a library of gene circuits comprising all possible link combinations in a simplified Rb–E2F network. We identified a minimal circuit that is able to generate robust, resettable bistability. This minimal circuit contains a feed-forward loop coupled with a mutual-inhibition feedback loop, which forms an AND-gate control of the E2F activation. Underscoring its importance, experimental disruption of this circuit abolishes maintenance of the activated E2F state, supporting its importance for the bistability of the Rb–E2F system. Our findings suggested basic design principles for the robust control of the bistable cell cycle entry at the R-point.
doi:10.1038/msb.2011.19
PMCID: PMC3101952  PMID: 21525871
bistable switch; cell cycle checkpoint; design principle; Rb–E2F pathway; robustness
12.  Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics 
PLoS Biology  2009;7(10):e1000213.
Gene networks are an efficient route for associating candidate genes with biological processes. Here, networks are used to discover more than 15 new genes for ribosomal subunit maturation, rRNA processing, and ribosomal export from the nucleus.
Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.
Author Summary
Ribosomes are the extremely complex cellular machines responsible for constructing new proteins. In eukaryotic cells, such as yeast, each ribosome contains more than 80 protein or RNA components. These complex machines must themselves be assembled by an even more complex machinery spanning multiple cellular compartments and involving perhaps 200 components in an ordered series of processing events, resulting in delivery of the two halves of the mature ribosome, the 40S and 60S components, to the cytoplasm. The ribosome biogenesis machinery has been only partially characterized, and many lines of evidence suggest that there are additional components that are still unknown. We employed an emerging computational technique called network-guided genetics to identify new candidate genes for this pathway. We then tested the candidates in a battery of experimental assays to determine what roles the genes might play in the biogenesis of ribosomes. This approach proved an efficient route to the discovery of new genes involved in ribosome biogenesis, significantly extending our understanding of a universally conserved eukaryotic process.
doi:10.1371/journal.pbio.1000213
PMCID: PMC2749941  PMID: 19806183
13.  Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity 
PLoS Pathogens  2012;8(3):e1002612.
Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
Author Summary
Retroviruses are intracellular parasites that utilize the host translation machinery to catalyze viral protein synthesis. The activity of the translation machinery fluctuates during cell cycle progression and is reduced in the G2/M phase. HIV-1 infection causes the cells to arrest in the G2/M phase, which has the potential to alter the activity of the translation machinery. Herein several lines of evidence demonstrated that lymphocyte mRNA translation is suppressed by the action of HIV-1 accessory protein Vpr. The molecular basis of translation suppression is reduced activity of the rate-limiting translation intitation factor, eIF4E. However, synthesis of the viral structural proteins is sustained and is due to the difference in composition of the viral and cellular mRNA-ribonucleoprotein complexes. Both cellular and completely spliced viral mRNAs are predominantly associated with the cytoplasmic cap binding protein, eIF4E. In contrast, unspliced HIV-1 mRNAs are predominantly associated with the components of the nuclear cap binding complex (CBC). The retention of CBC on the viral mRNAs provides a mechanism to sustain viral protein synthesis. This newly characterized interface of the virus-host-protein synthesis machinery is likely a cellular adaptation used to enable synthesis of proteins that reengage the cell cycle and facilitate recovery from stress.
doi:10.1371/journal.ppat.1002612
PMCID: PMC3310836  PMID: 22457629
14.  Attenuation of miR-126 Activity Expands HSC In Vivo without Exhaustion 
Cell Stem Cell  2012;11(6):799-811.
Summary
Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.
Graphical Abstract
Highlights
► miR-126 is a novel regulator of the HSC quiescence/proliferation equilibrium ► Reduction in miR-126 induces an expansion of long-term HSC without exhaustion ► Constitutive miR-126 expression promotes HSC quiescence and progenitor proliferation ► miR-126 attenuates PI3K/AKT activation in response to cytokine stimulation
miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway to promote HSC quiescence and progenitor proliferation.
doi:10.1016/j.stem.2012.09.001
PMCID: PMC3517970  PMID: 23142521
15.  Stochastic E2F Activation and Reconciliation of Phenomenological Cell-Cycle Models 
PLoS Biology  2010;8(9):e1000488.
A new, stochastic model of entry into the mammalian cell cycle provides a mechanistic understanding of the temporal variability observed across populations of cells and reconciles previously proposed phenomenological cell-cycle models.
The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.
Author Summary
Mammalian cells enter the division cycle in response to appropriate growth signals. For each cell, the decision to do so is critically dependent on the interplay between environmental cues and the internal state of the cell and is influenced by random fluctuations in cellular processes. Indeed, experimental evidence indicates that cell cycle entry is highly variable from cell to cell, even within a clonal population. To account for such variability, a number of phenomenological models have been previously proposed. These models primarily fall into two types depending on their fundamental assumptions on the origin of the variability. “Transition probability” models presume that variability in cell cycle entry originates from the fact that entry in each individual cell is random but also governed by a fixed probability. In contrast, “growth-controlled” models assume that the growth rates across a population are variable and result in cells that are out of phase developmentally. While both kinds of models provide a good fit to experimental data, their lack of mechanistic details limits their predictive power and has led to unresolved debate between their practitioners. In this study, we developed a mechanistically based stochastic model of the temporal dynamics of activation of the E2F transcription factor, which is used here as a marker of the transition of cells from quiescence to active cell cycling. Using this model, we show that “transition probability” and “growth-controlled” models can be reconciled by incorporation of a small number of basic cellular parameters related to protein synthesis and turnover, protein modification, stochasticity, and the like. Essentially our work shows that each kind of phenomenological model holds true for describing a particular aspect of the cell cycle transition. We suggest that incorporation of basic cellular parameters in this manner into phenomenological models may constitute a broadly applicable approach to defining concise, quantitative phenotypes of cell physiology.
doi:10.1371/journal.pbio.1000488
PMCID: PMC2943438  PMID: 20877711
16.  The Small Subunit Processome Is Required for Cell Cycle Progression at G1 
Molecular Biology of the Cell  2004;15(11):5038-5046.
Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.
doi:10.1091/mbc.E04-06-0515
PMCID: PMC524768  PMID: 15356263
17.  The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation 
PLoS Genetics  2015;11(5):e1005233.
Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5’cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3’ UTR motif. Caf20p binds all tested motif-containing 3’ UTRs. Caf20p and the 3’UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3’UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.
Author Summary
In eukaryotic cells protein synthesis initiation factor eIF4E controls mRNA selection by interacting with the mRNA 5’ cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment and repress translation of their target mRNAs. The yeast Saccharomyces cerevisiae has two 4E-BPs Caf20p and Eap1p that regulate distinct but overlapping sets of mRNAs. Here, we describe genome wide experiments to identify protein and RNA partners of each 4E-BP, with a greater focus on Caf20p. We present evidence that the 4E-BPs unexpectedly bind to ribosomes, an interaction that is not dependent on eIF4E binding. We also define a core set of over 500 Caf20p target mRNAs that fall into two classes with distinct features. One mRNA class, representing 25% of the targets, binds Caf20p independently of its eIF4E interaction and instead via a novel 3’ UTR interaction. Our data indicate that these proteins can repress mRNA-specific protein synthesis independently of their known role as eIF4E-binding proteins.
doi:10.1371/journal.pgen.1005233
PMCID: PMC4431810  PMID: 25973932
18.  Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control 
Combining translating ribosome affinity purification with RNA-seq for cell-specific profiling of translating RNAs in developing flowers.Cell type comparisons of cell type-specific hormone responses, promoter motifs, coexpressed cognate binding factor candidates, and splicing isoforms.Widespread post-transcriptional regulation at both the intron splicing and translational stages.A new class of noncoding RNAs associated with polysomes.
What constitutes a differentiated cell type? How much do cell types differ in their transcription of genes? The development and functions of tissues rely on constant interactions among distinct and nonequivalent cell types. Answering these questions will require quantitative information on transcriptomes, proteomes, protein–protein interactions, protein–nucleic acid interactions, and metabolomes at cellular resolution. The systems approaches emerging in biology promise to explain properties of biological systems based on genome-wide measurements of expression, interaction, regulation, and metabolism. To facilitate a systems approach, it is essential first to capture such components in a global manner, ideally at cellular resolution.
Recently, microarray analysis of transcriptomes has been extended to a cellular level of resolution by using laser microdissection or fluorescence-activated sorting (for review, see Nelson et al, 2008). These methods have been limited by stresses associated with cellular separation and isolation procedures, and biases associated with mandatory RNA amplification steps. A newly developed method, translating ribosome affinity purification (TRAP; Zanetti et al, 2005; Heiman et al, 2008; Mustroph et al, 2009), circumvents these problems by epitopetagging a ribosomal protein in specific cellular domains to selectively purify polysomes. We combined TRAP with deep sequencing, which we term TRAP-seq, to provide cell-level spatiotemporal maps for Arabidopsis early floral development at single-base resolution.
Flower development in Arabidopsis has been studied extensively and is one of the best understood aspects of plant development (for review, see Krizek and Fletcher, 2005). Genetic analysis of homeotic mutants established the ABC model, in which three classes of regulatory genes, A, B and C, work in a combinatorial manner to confer organ identities of four whorls (Coen and Meyerowitz, 1991). Each class of regulatory gene is expressed in a specific and evolutionarily conserved domain, and the action of the class A, B and C genes is necessary for specification of organ identity (Figure 1A).
Using TRAP-seq, we purified cell-specific translating mRNA populations, which we and others call the translatome, from the A, B and C domains of early developing flowers, in which floral patterning and the specification of floral organs is established. To achieve temporal specificity, we used a floral induction system to facilitate collection of early stage flowers (Wellmer et al, 2006). The combination of TRAP-seq with domain-specific promoters and this floral induction system enabled fine spatiotemporal isolation of translating mRNA in specific cellular domains, and at specific developmental stages.
Multiple lines of evidence confirmed the specificity of this approach, including detecting the expression in expected domains but not in other domains for well-studied flower marker genes and known physiological functions (Figures 1B–D and 2A–C). Furthermore, we provide numerous examples from flower development in which a spatiotemporal map of rigorously comparable cell-specific translatomes makes possible new views of the properties of cell domains not evident in data obtained from whole organs or tissues, including patterns of transcription and cis-regulation, new physiological differences among cell domains and between flower stages, putative hormone-active centers, and splicing events specific for flower domains (Figure 2A–D). Such findings may provide new targets for reverse genetics studies and may aid in the formulation and validation of interaction and pathway networks.
Beside cellular heterogeneity, the transcriptome is regulated at several steps through the life of mRNA molecules, which are not directly available through traditional transcriptome profiling of total mRNA abundance. By comparing the translatome and transcriptome, we integratively profiled two key posttranscriptional control points, intron splicing and translation state. From our translatome-wide profiling, we (i) confirmed that both posttranscriptional regulation control points were used by a large portion of the transcriptome; (ii) identified a number of cis-acting features within the coding or noncoding sequences that correlate with splicing or translation state; and (iii) revealed correlation between each regulation mechanism and gene function. Our transcriptome-wide surveys have highlighted target genes transcripts of which are probably under extensive posttranscriptional regulation during flower development.
Finally, we reported the finding of a large number of polysome-associated ncRNAs. About one-third of all annotated ncRNA in the Arabidopsis genome were observed co-purified with polysomes. Coding capacity analysis confirmed that most of them are real ncRNA without conserved ORFs. The group of polysome-associated ncRNA reported in this study is a potential new addition to the expanding riboregulator catalog; they could have roles in translational regulation during early flower development.
Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.
doi:10.1038/msb.2010.76
PMCID: PMC2990639  PMID: 20924354
Arabidopsis; flower; intron; transcriptome; translation
19.  eIF6 over-expression increases the motility and invasiveness of cancer cells by modulating the expression of a critical subset of membrane-bound proteins 
BMC Cancer  2015;15:131.
Background
Eukaryotic Initiation factor 6 (eIF6) is a peculiar translation initiation factor that binds to the large 60S ribosomal subunits, controlling translation initiation and participating in ribosome biogenesis. In the past, knowledge about the mechanisms adopted by the cells for controlling protein synthesis by extracellular stimuli has focused on two translation initiation factors (eIF4E and eIF2), however, recent data suggest eIF6 as a newcomer in the control of downstream of signal transduction pathways. eIF6 is over-expressed in tumors and its decreased expression renders cells less prone to tumor growth. A previous work from our laboratory has disclosed that over-expression of eIF6 in transformed cell lines markedly increased cell migration and invasion.
Methods
Here, we performed a quantitative proteomic analysis of membrane-associated proteins in A2780 ovarian cancer cells over-expressing eIF6. Differentially expressed proteins upon eIF6 overproduction were further investigated in silico by Ingenuity Pathway Analysis (IPA). RT-qPCR and Western blot were performed in order to validate the proteomic data. Furthermore, the effects of a potent and selective inhibitor ML-141 in A2780 cells were evaluated using transwell migration assay. Finally, we explored the effects of eIF6 over-expression on WM793 primary melanoma cell lines.
Results
We demonstrated that: (i) the genes up-regulated upon eIF6 overproduction mapped to a functional network corresponding to cellular movements in a highly significant way; (ii) cdc42 plays a pivotal role as an effector of enhanced migratory phenotype induced upon eIF6 over-expression; (iii) the variations in abundance observed for cdc42 protein occur at a post-transcriptional level; (iv) the increased cell migration/invasion upon eIF6 over-expression was generalizable to other cell line models.
Conclusions
Collectively, our data confirm and further extend the role of eIF6 in enhancing cell migration/invasion. We show that a number of membrane-associated proteins indeed vary in abundance upon eIF6 over-expression, and that the up-regulated proteins can be located within a functional network controlling cell motility and tumor metastasis. Full understanding of the role eIF6 plays in the metastatic process is important, also in view of the fact that this factor is a potentially druggable target to be exploited for new anti-cancer therapies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1106-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1106-3
PMCID: PMC4381359  PMID: 25886394
Protein synthesis; Ribosome biogenesis; eIF6; cdc42; Cell migration
20.  A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation 
PLoS Pathogens  2016;12(1):e1005379.
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
Author Summary
Norovirus infections cause acute gastroenteritis and are a growing worldwide problem in human health. A critical early step in infection is translation of the viral RNA genome to produce the proteins needed to assemble new virus particles. In mouse noroviruses (MNV), which provide a useful model for studying human noroviruses, the VPg protein attached to the viral RNA is essential for this process because it interacts with a cellular protein, eIF4G, that is normally involved in initiating protein synthesis from the messenger RNA of host genes. We have used a variety of biochemical and biophysical experiments to measure how well MNV VPg binds to eIF4G and to identify the parts of both proteins that are involved in this interaction. We show that a sequence of about 20 amino acids at one end of MNV VPg–the C terminus– allows it to bind to a well-defined domain within eIF4G (called HEAT-1), and that it may adopt a helical structure when doing so. Our data suggest that this interaction is common to all noroviruses, including types that infect humans. We have also shown that the MNV VPg C-terminal peptide can inhibit norovirus protein synthesis, which raises the possibility that the VPg-eIF4G interaction could be targeted in the design of antiviral drugs.
doi:10.1371/journal.ppat.1005379
PMCID: PMC4703368  PMID: 26734730
21.  Inhibition of eIF2α dephosphorylation inhibits ErbB2-induced deregulation of mammary acinar morphogenesis 
BMC Cell Biology  2009;10:64.
Background
The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in ~30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2α inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2α can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2α phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis.
Results
We tested whether ErbB2 signaling influenced eIF2α signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt) Neu/ErbB2 or a constitutively active (CA) variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2α signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2α or induction of its downstream target ATF4. However, inhibition of eIF2α dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells.
Conclusion
Depending on the strength of ErbB2 signaling there is a differential regulation of CHOP and eIF2α phosphorylation. ErbB2 uncouples in basal conditions eIF2α phosphorylation from CHOP induction. However, this signal was restored by salubrinal treatment in Wt-ErbB2 expressing MCF10A cells as these DCIS-like structures underwent luminal clearing. In CA-ErbB2 structures apoptosis is not induced by salubrinal and instead a state of quiescence with reduced proliferation was achieved. Treatments that stabilize P-eIF2α levels may be effective in treating ErbB2 positive cancers without severely disrupting normal tissue function and structure.
doi:10.1186/1471-2121-10-64
PMCID: PMC2754445  PMID: 19754954
22.  Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing 
PLoS Pathogens  2013;9(6):e1003460.
Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.
Author Summary
Influenza A virus is a major human pathogen, which causes approximately 500,000 deaths/year worldwide. In pandemic years, influenza infection can lead to even higher mortality rates, as in 1918, when ∼30–50 million deaths occurred worldwide. In this manuscript, we identified a novel function for the cellular protein termed NS1-BP as a regulator of the influenza A virus life cycle. We found that NS1-BP, together with other host factors, mediates the expression of a key viral protein termed M2. NS1-BP and its interacting partner hnRNP K specifically regulate alternative splicing of the viral M1 mRNA segment, which generates the M2 mRNA that is translated into the essential viral M2 protein. The M2 protein is key for viral uncoating and entry into the host cell cytoplasm. Altogether, inhibition of NS1-BP and hnRNP K functions regulate influenza A virus gene expression and replication. In sum, these studies revealed new functions for the cellular proteins NS1-BP and hnRNP K during viral RNA expression, which facilitate the influenza A virus life cycle.
doi:10.1371/journal.ppat.1003460
PMCID: PMC3694860  PMID: 23825951
23.  Integrating Transcriptomic and Proteomic Data Using Predictive Regulatory Network Models of Host Response to Pathogens 
PLoS Computational Biology  2016;12(7):e1005013.
Mammalian host response to pathogenic infections is controlled by a complex regulatory network connecting regulatory proteins such as transcription factors and signaling proteins to target genes. An important challenge in infectious disease research is to understand molecular similarities and differences in mammalian host response to diverse sets of pathogens. Recently, systems biology studies have produced rich collections of omic profiles measuring host response to infectious agents such as influenza viruses at multiple levels. To gain a comprehensive understanding of the regulatory network driving host response to multiple infectious agents, we integrated host transcriptomes and proteomes using a network-based approach. Our approach combines expression-based regulatory network inference, structured-sparsity based regression, and network information flow to infer putative physical regulatory programs for expression modules. We applied our approach to identify regulatory networks, modules and subnetworks that drive host response to multiple influenza infections. The inferred regulatory network and modules are significantly enriched for known pathways of immune response and implicate apoptosis, splicing, and interferon signaling processes in the differential response of viral infections of different pathogenicities. We used the learned network to prioritize regulators and study virus and time-point specific networks. RNAi-based knockdown of predicted regulators had significant impact on viral replication and include several previously unknown regulators. Taken together, our integrated analysis identified novel module level patterns that capture strain and pathogenicity-specific patterns of expression and helped identify important regulators of host response to influenza infection.
Author Summary
An important challenge in infectious disease research is to understand how the human immune system responds to different types of pathogenic infections. An important component of mounting proper response is the transcriptional regulatory network that specifies the context-specific gene expression program in the host cell. However, our understanding of this regulatory network and how it drives context-specific transcriptional programs is incomplete. To address this gap, we performed a network-based analysis of host response to influenza viruses that integrated high-throughput mRNA- and protein measurements and protein-protein interaction networks to identify virus and pathogenicity-specific modules and their upstream physical regulatory programs. We inferred regulatory networks for human cell line and mouse host systems, which recapitulated several known regulators and pathways of the immune response and viral life cycle. We used the networks to study time point and strain-specific subnetworks and to prioritize important regulators of host response. We predicted several novel regulators, both at the mRNA and protein levels, and experimentally verified their role in the virus life cycle based on their ability to significantly impact viral replication.
doi:10.1371/journal.pcbi.1005013
PMCID: PMC4942116  PMID: 27403523
24.  Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries 
mBio  2016;7(2):e00029-16.
ABSTRACT
Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.
IMPORTANCE
Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.
doi:10.1128/mBio.00029-16
PMCID: PMC4807356  PMID: 27025248
25.  Deficiency in spliceosome-associated factor CTNNBL1 does not affect ongoing cell cycling but delays exit from quiescence and results in embryonic lethality in mice 
Cell Cycle  2013;12(5):732-742.
CTNNBL1 is an armadillo-repeat protein that associates with the CDC5L/Prp19 complex of the spliceosome. Unlike the majority of spliceosomal proteins (and despite having no obvious homologs), CTNNBL1 is inessential for cell viability as revealed by studies in both vertebrate B cell lines and in fission yeast. Here, however, we show that ablation of CTNNBL1 in the mouse germline results in mid-gestation embryonic lethality but that lineage-specific CTNNBL1 ablation in early B cell precursors does not affect the production and abundance of mature B lymphocytes. However, CTNNBL1-deficient resting B lymphocytes show sluggish exit from quiescence on cell activation, although once entry into cycle has initiated, proliferation and differentiation in response to mitogenic stimuli continue largely unaffected. A similar sluggish exit from quiescence is also observed on reprovision of nutrients to nitrogen-starved CTNNBL1-deficient yeast. The results indicate that, whereas other RNA splicing-associated factors have been connected to cell cycle progression, CTNNBL1 plays no essential role in cycling cells but does fulfill an evolutionarily conserved function in helping cells to undergo efficient exit from quiescence following activation.
doi:10.4161/cc.23594
PMCID: PMC3610721  PMID: 23343763
CTNNBL1; cell cycle; quiescence; RNA splicing; B lymphocyte; yeast; conditional knockout mice

Results 1-25 (1509444)