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Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.
Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.
Mitochondrial translation is essentially bacteria-like, reflecting the bacterial endosymbiotic ancestry of the eukaryotic organelle. However, unlike the translation system of its bacterial ancestors, mitochondrial translation is limited to just a few mRNAs, mainly coding for components of the respiratory complex. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in bacteria, but only IF2 is universal in mitochondria (mIF2). We analyse the distribution of mitochondrial translation initiation factors and their sequence features, given two well-propagated claims: first, a sequence insertion in mitochondrial IF2 (mIF2) compensates for the universal lack of IF1 in mitochondria, and secondly, no homologue of mitochondrial IF3 (mIF3) is identifiable in Saccharomyces cerevisiae. Our comparative sequence analysis shows that, in fact, the mIF2 insertion is highly variable and restricted in length and primary sequence conservation to vertebrates, while phylogenetic and in vivo complementation analyses reveal that an uncharacterized S. cerevisiae mitochondrial protein currently named Aim23p is a bona fide evolutionary and functional orthologue of mIF3. Our results highlight the lineage-specific nature of mitochondrial translation and emphasise that comparative analyses among diverse taxa are essential for understanding whether generalizations from model organisms can be made across eukaryotes.
Peptide-receptor signaling is an important system for intercellular communication, regulating many developmental processes. A single process can be controlled by several distinct signaling peptides. However, since peptide-receptor modules are usually studied separately, their mechanistic interactions remain largely unexplored. Two phylogenetically unrelated peptide-receptor modules, GLV6/GLV10-RGI and TOLS2/PIP2-RLK7, independently described as inhibitors of lateral root initiation, show striking similarities between their expression patterns and gain- and loss-of-function phenotypes, suggesting a common function during lateral root spacing and initiation. The GLV6/GLV10-RGI and TOLS2/PIP2-RLK7 modules trigger similar transcriptional changes, likely in part via WRKY transcription factors. Their overlapping set of response genes includes PUCHI and PLT5, both required for the effect of GLV6/10, as well as TOLS2, on lateral root initiation. Furthermore, both modules require the activity of MPK6 and can independently trigger MPK3/MPK6 phosphorylation. The GLV6/10 and TOLS2/PIP2 signaling pathways seem to converge in the activation of MPK3/MPK6, leading to the induction of a similar transcriptional response in the same target cells, thereby regulating lateral root initiation through a (partially) common mechanism. Convergence of signaling pathways downstream of phylogenetically unrelated peptide-receptor modules adds an additional, and hitherto unrecognized, level of complexity to intercellular communication networks in plants.
A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.
After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.
Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.
Photosynthetic CO2 assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO2-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.
The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRRs, LlRR1, LlRR2, LlRR10, LlRR11 and LlRR12, in L. lancifolium for the first time. The five LlRRs encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRRs are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.
Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.
In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.
Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A-deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A-deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.
Translation initiation using noncanonical initiator substrates with poor peptidyl donor activities, such as N-acetyl-l-proline (AcPro), induces the N-terminal drop-off-reinitiation event. Thereby, the initiator tRNA drops-off from the ribosome and the translation reinitiates from the second amino acid to yield a truncated peptide lacking the N-terminal initiator substrate. In order to suppress this event for the synthesis of full-length peptides, here we have devised a chimeric initiator tRNA, referred to as tRNAiniP, whose D-arm comprises a recognition motif for EF-P, an elongation factor that accelerates peptide bond formation. We have shown that the use of tRNAiniP and EF-P enhances the incorporation of not only AcPro but also d-amino, β-amino and γ-amino acids at the N-terminus. By optimizing the translation conditions, e.g. concentrations of translation factors, codon sequence and Shine-Dalgarno sequence, we could achieve complete suppression of the N-terminal drop-off-reinitiation for the exotic amino acids and enhance the expression level of full-length peptide up to 1000-fold compared with the use of the ordinary translation conditions.
Cold-stress in Escherichia coli induces de novo synthesis of translation initiation factors IF1, IF2 and IF3 while ribosome synthesis and assembly slow down. Consequently, the IFs/ribosome stoichiometric ratio increases about 3-fold during the first hours of cold adaptation. The IF1 and IF3 increase plays a role in translation regulation at low temperature (cold-shock-induced translational bias) but so far no specific role could be attributed to the extra copies of IF2. In this work, we show that the extra-copies of IF2 made after cold stress are associated with immature ribosomal subunits together with at least another nine proteins involved in assembly and/or maturation of ribosomal subunits. This finding, coupled with evidence that IF2 is endowed with GTPase-associated chaperone activity that promotes refolding of denatured GFP, and the finding that two cold-sensitive IF2 mutations cause the accumulation of immature ribosomal particles, indicate that IF2 is yet another GTPase protein that participates in ribosome assembly/maturation, especially at low temperatures. Overall, these findings are instrumental in redefining the functional role of IF2, which cannot be regarded as being restricted to its well documented functions in translation initiation of bacterial mRNA.
The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.
The genetic diversity of the influenza virus hinders the use of broad spectrum antiviral drugs and favors the appearance of resistant strains. Single-stranded DNA aptamers represent an innovative approach with potential application as antiviral compounds. The mRNAs of influenza virus possess a 5'cap structure and a 3'poly(A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins. We have studied the potential of two specific aptamers that recognize PABP1 (ApPABP7 and ApPABP11) to act as anti-influenza drugs. Both aptamers reduce viral genome expression and the production of infective influenza virus particles. The interaction of viral polymerase with the eIF4GI translation initiation factor is hindered by transfection of infected cells with both PABP1 aptamers, and ApPABP11 also inhibits the association of NS1 with PABP1 and eIF4GI. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes.
Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.
Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.
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