Enforcing temporal control of maternal mRNA translation during oocyte cell‐cycle progression

Meiotic cell‐cycle progression in progesterone‐stimulated Xenopus oocytes requires that the translation of pre‐existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3′ untranslated region (3′ UTR), which respond to cell cycle‐dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE does not direct early mRNA translation. Rather, early translation is directed through specific early factors, including the Musashi‐binding element (MBE) and the MBE‐binding protein, Musashi. Our findings indicate that although the cyclin B5 3′ UTR contains both CPEs and an MBE, the MBE is the critical regulator of early translation. The cyclin B2 3′ UTR contains CPEs, but lacks an MBE and is translationally activated late in maturation. Finally, utilizing antisense oligonucleotides to attenuate endogenous Musashi synthesis, we show that Musashi is critical for the initiation of early class mRNA translation and for the subsequent activation of CPE‐dependant mRNA translation.     

Cracking pre‐40S ribosomal subunit structure by systematic analyses of RNA–protein cross‐linking

Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre‐ribosomal particles. We report rRNA‐binding sites for six late‐acting 40S ribosome synthesis factors, three of which cluster around the 3′ end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ structure formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1‐ and Tsr1‐binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3′‐endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre‐rRNA. Nob1 binds before pre‐rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.     

The C domain of translation termination factor eRF1 is close to the stop codon in the A site of the 80S ribosome

The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome.     

IRES‐mediated translation of cofilin regulates axonal growth cone extension and turning

In neuronal development, dynamic rearrangement of actin promotes axonal growth cone extension, and spatiotemporal translation of local mRNAs in response to guidance cues directs axonal growth cone steering, where cofilin plays a critical role. While regulation of cofilin activity is well studied, regulatory mechanism for cofilin mRNA translation in neurons is unknown. In eukaryotic cells, proteins can be synthesized by cap‐dependent or cap‐independent mechanism via internal ribosome entry site (IRES)‐mediated translation. IRES‐mediated translation has been reported in various pathophysiological conditions, but its role in normal physiological environment is poorly understood. Here, we report that 5′UTR of cofilin mRNA contains an IRES element, and cofilin is predominantly translated by IRES‐mediated mechanism in neurons. Furthermore, we show that IRES‐mediated translation of cofilin is required for both axon extension and axonal growth cone steering. Our results provide new insights into the function of IRES‐mediated translation in neuronal development.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.     

Tpo1‐mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H₂O₂) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H₂O₂ tolerance. Thus, central components of the stress response are timed through Tpo1‐controlled polyamine export.     

Overexpression limits of fission yeast cell‐cycle regulators in vivo and in silico

Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic ‘tug‐of‐war’ (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell‐cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation‐initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase‐activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.     

Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro

We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 10⁵ and 24 × 10⁵. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 10³ and 320 × 10³. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose.     

Global microRNA level regulation of EGFR‐driven cell‐cycle protein network in breast cancer

The EGFR‐driven cell‐cycle pathway has been extensively studied due to its pivotal role in breast cancer proliferation and pathogenesis. Although several studies reported regulation of individual pathway components by microRNAs (miRNAs), little is known about how miRNAs coordinate the EGFR protein network on a global miRNA (miRNome) level. Here, we combined a large‐scale miRNA screening approach with a high‐throughput proteomic readout and network‐based data analysis to identify which miRNAs are involved, and to uncover potential regulatory patterns. Our results indicated that the regulation of proteins by miRNAs is dominated by the nucleotide matching mechanism between seed sequences of the miRNAs and 3′‐UTR of target genes. Furthermore, the novel network‐analysis methodology we developed implied the existence of consistent intrinsic regulatory patterns where miRNAs simultaneously co‐regulate several proteins acting in the same functional module. Finally, our approach led us to identify and validate three miRNAs (miR‐124, miR‐147 and miR‐193a‐3p) as novel tumor suppressors that co‐target EGFR‐driven cell‐cycle network proteins and inhibit cell‐cycle progression and proliferation in breast cancer.     

The iron–sulphur protein RNase L inhibitor functions in translation termination

The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.     

Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint

Cell division cycle 5‐like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre‐messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell‐cycle checkpoint kinase ataxia‐telangiectasia and Rad3‐related (ATR). Depletion of Cdc5L by RNA‐mediated interference methods results in a defective S‐phase cell‐cycle checkpoint and cellular sensitivity in response to replication‐fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR‐binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L‐depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR‐mediated cell‐cycle checkpoint in response to genotoxic agents.     

Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non‐selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy‐dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2‐type ribonuclease, generating 3′‐NMPs that were immediately converted to nucleosides by the vacuolar non‐specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re‐assimilated, but excreted from the cell. Bulk non‐selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.     

Interleukin-1 receptor-like proteins synthesized by translation, in vitro, of immunopurified polysomal messenger RNA

Interleukin-1 (IL-1) receptors can be solubilized from murine cell surfaces and immunoprecipitated with a xenogeneic rat antiserum raised in this laboratory. We demonstrated first that this antiserum contains antibodies directed against IL-1 receptors. We have now successfully used this antiserum as a reagent to immunopurify polysomes along with their messenger RNA from a murine leukemic cell line known to express relatively high levels of IL-1 receptors. The immunoselected mRNA was translated into proteins in vitro. The translation products contained an IL-1 binding protein which could specifically bind to immobilized IL-1 but not to other immobilized ligands such as interleukin-2 or tumor necrosis factor-alpha. The translation products which bound to IL-1 could be acid-eluted from the immobilized ligand, and the proteins released could still specifically bind to IL-1 in a receptor-ligand binding reaction. The eluted IL-1 binding proteins, as well as soluble receptor-ligand complexes derived from them, could also be immunoprecipitated with the xenogeneic rat antiserum. The xenogeneic rat antiserum could, furthermore, immunoprecipitate the IL-1 binding proteins from the translated products before ligand was added. The residual translated products no longer interacted with IL-1. We conclude that our antiserum contains antibodies that recognize determinants expressed on the following proteins: on nascent chains of IL-1 binding proteins; on soluble translated IL-1 binding proteins; on soluble complexes of IL-1 binding proteins that had been cross-linked with IL-1 ligand; and on cell surface-associated IL-1 receptors. The translated and unprocessed IL-1 binding proteins have a molecular mass of approximately 52,000-56,000 daltons.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

Longevity and resistance to cold stress in cold‐stress selected lines and their controls in Drosophila melanogaster

Thermal environments can influence many fitness‐related traits including life span. Here, we assess whether longevity in Drosophila melanogaster can experimentally evolve as a correlated response to cold‐stress selection, and whether genotype‐by‐temperature and sex‐by‐temperature interactions are significant components of variation in life span. Three replicated S lines were cold‐stress selected and compared with their respective unselected controls (Clines) in the 16^th generation of thermal selection. Cold‐stress resistance exhibited a substantial direct response to selection, and also showed a significant interaction between sex and type of line. Mean longevity exhibited a significant interaction between adult test temperature (14 and 25 °C) and line (with suggestive evidence for increased longevity of S lines when tested at 14 °C), but there was no evidence for increased longevity in S lines at normal temperatures (i.e. 25 °C). Another temperature‐dependent effect was sex‐specific, with males being the longer lived sex at 25 °C but the less long‐lived sex at 14 °C. Additionally, we tested in an exploratory way the relationship between longevity and cold‐stress resistance by also measuring resistance to a prefreezing temperature before and after one generation of longevity selection at 14 °C (selection intensity, i = 1.47 for S lines, and 1.42 for C lines). In this longevity selection, we found that cold‐stress resistance increased by about 6% in S lines and 18% in C lines. However, taken together, the results indicate no simple relationship between longevity and cold‐stress resistance, with genotype‐by‐sex interactions in both traits. Temperature dependent interaction in longevity is apparent between S and C lines, and sex‐specific variation in mean longevity also depends on temperature.