YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5′UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.     

Transient MPK6 activation in response to oxygen deprivation and reoxygenation is mediated by mitochondria and aids seedling survival in Arabidopsis

Mitogen-activated protein kinases (MPKs) are regulated by diverse stresses with a reactive oxygen species (ROS) component. Here, we report the rapid and transient activation of MPK3, MPK4 and MPK6 upon oxygen deprivation as well as reoxygenation in seedlings of Arabidopsis thaliana. MPK activation peaked within 2 h of oxygen deprivation and again at a higher magnitude within 5 min of reoxygenation. MPK6 was the predominant kinase regulated by oxygen availability in both aerial and root tissue, except in mpk6 mutants, which displayed compensatory activation of MPK3. A universal consequence of oxygen deprivation in eukaryotes is inhibition of the terminal step of the mitochondrial electron transport chain (mETC). We demonstrate that treatment of seedlings with the mETC inhibitors antimycin A and potassium cyanide under normoxia promotes transient MPK6 and MPK3 activation. Confocal imaging of seedlings provided evidence that both oxygen deprivation and mETC inhibitors stimulate mitochondria-associated ROS production. We found that seedling survival of prolonged oxygen deprivation was improved in transgenics that ectopically overexpress MPK3, MPK4 and MPK6, but the induction of mRNAs associated with low oxygen acclimation responses were not markedly altered in MPK6 overexpression lines or mpk6 loss-of-function mutants. However, distinctions in MPK6 activation potential were correlated with other differences in mRNAs accumulation. Our findings suggest that oxygen deprivation and reoxygenation trigger mitochondrial ROS production to activate MPK signaling, which in turn regulate reversible processes that aid survival of transient oxygen deprivation.     

Glucose promotes caspase-dependent delayed cell death after a transient episode of oxygen and glucose deprivation in SH-SY5Y cells

Brain ischemia causes neuronal cell death by several mechanisms involving necrotic and apoptotic processes. The contributions of each process depend on conditions such as the severity and duration of ischemia, and the availability of ATP. We examined whether glucose affected the development of apoptosis after transient ischemia, and whether this was sensitive to caspase inhibition. Retinoic acid-differentiated SH-SY5Y human neuroblastoma cells were subjected to oxygen and glucose deprivation for 15 h followed by various periods of reoxygenation in either the presence or absence of glucose. Oxygen and glucose deprivation induced cell death in the hours following reoxygenation, as detected by propidium iodide staining. At the end of the period of oxygen and glucose deprivation, both cytochrome c and apoptosis-inducing factor translocated from mitochondria to cytosol. Reoxygenation in the presence of glucose accelerated cell death, and enhanced caspase-3 activity and apoptosis. The glucose-dependent increase in apoptosis was prevented by treatment with the caspase inhibitor zVAD-fmk, but not with calpeptin, a calpain inhibitor. Nevertheless, both zVAD-fmk and calpeptin decreased cell death in the glucose-treated group. ATP levels dropped dramatically after oxygen and glucose deprivation, but recovered steadily thereafter, and were significantly higher at 6 h of reoxygenation in the glucose-treated group. This indicates that energy recovery may promote the glucose-dependent cell death. We conclude that glucose favours the development of caspase-dependent apoptosis during reoxygenation following oxygen and glucose deprivation.     

Mouse kidney nonpolysomal messenger ribonucleic acid: metabolism, coding function, and translational activity

To elucidate the distribution and function of mRNA in mouse kidney cytoplasm, we compared mRNA isolated from polysomal (greater than 80S) and native postpolysomal (20--80S) ribonucleoproteins with respect to synthesis and lifetime, sequence content, and translational activity. The 20--25% of cytoplasmic mRNA recovered from postpolysomal ribonucleoprotein is similar to polysomal mRNA in size (20--22S), in apparent half-life (11--13 h), in major products of cell-free translation, and in nucleotide complexity (approximately 4 x 10(7) nucleotides). The labeling kinetics of polysomal and postpolysomal mRNA suggest these mRNA populations are in equilibrium. [3H]cDNAs transcribed from polysomal and from postpolysomal poly(A)-containing mRNAs react with template mRNA and with the heterologous mRNA at the same rate (Cot1/2 approximately 6.3 mol.s/L) and to the same extent (95%). Therefore, these mRNAs are equally diverse and homologous and occur at similar relative frequencies. Postpolysomal mRNA directs cell-free protein synthesis at only approximately 30% of the rate of polysomal mRNA and to only 30% of the extent of mRNA from polysomes. Postpolysomal mRNA is approximately 3-fold less sensitive than polysomal mRNA to inhibition of translation by m7GMP, suggesting postpolysomal mRNA contains a greater fraction of molecules deficient in 5'-terminal caps. Postpolysomal mRNA may derive from renal mRNAs that initiate translation inefficiently and thus accumulate as postpolysomal ribonucleoproteins.     

Processing bodies require RNA for assembly and contain nontranslating mRNAs

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.     

Translation of reovirus messenger ribonucleic acids synthesized in vitro into reovirus proteins in a mouse L cell extract

Ten species of reovirus mRNAs were synthesized by incubating ATP, CTP, GTP, and UTP with reovirus particles which had been treated with chymotrypsin. The mRNAs obtained promote the synthesis of seven or more proteins in a cell-free system prepared from mouse L fibroblasts and the mobilities of these proteins during electrophoresis through polyacrylamide gels are indistinguishable from those of reo capsid proteins. Three antisera were prepared in rabbits: the first against the large size class of reo virion proteins, the second against the medium, and the third against the small. From the proteins whose synthesis was directed in the cell-free system by reo mRNAs each antiserum precipitates only those which correspond in size to the virion proteins against which the antiserum was prepared. The translation of reo mRNA occurs on large polysomal structures. Translation of peptide chains is initiated in the reo mRNA-directed cell-free system for at least 30 min. The average half-life of the various reo mRNAs during protein synthesis in our system is about 15 min. The optimal ionic conditions for reo mRNA translation are very different from those for encephalomyocarditis virus mRNA translation.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Change of Beclin‐1 dependent on ATP, [Ca2+]i and MMP in PC12 cells following oxygen‐glucose deprivation‐reoxygenation injury

Autophagy is usually up‐regulated to provide more ATP in response to starvation or OGD (oxygen‐glucose deprivation), but the relationship between autophagy and ATP, [Ca²⁺]_i (intracellular free Ca²⁺ concentration) or MMP (mitochondrial membrane potential) during reoxygenation is not yet fully clear. The role of autophagy is unknown in PC12 cells subjected to 2 h OGD with different time points of reoxygenation. In the present study, we showed that Beclin‐1 was up‐regulated beginning at 0 h reoxygenation peaking at 24 h and lasting for 48 h. Cell viability was decreased from 0 to 48 h reoxygenation, reaching its minimum at 10 h reoxygenation. ATP was decreased from 0 to 10 h reoxygenation, reaching its minimum at 4 h reoxygenation. A significant negative correlation was observed between ATP and Beclin‐1 (r = ?0.61, P<0.05) at 0 h reoxygenation, but ATP was not significant related (r = 0.24, P>0.05) to Beclin‐1 at 24 h reoxygenation. Besides, Nimodipine, a calcium antagonist, significantly reduced [Ca²⁺]_i and Beclin‐1, but increased MMP in OGD/R‐treated cells. At 24 h reoxygenation, Beclin‐1 expression reached its maximum, cell viability continued to increase, and ATP was higher than that before OGD. These results suggest that energy metabolism dysfunction can induce autophagy during OGD in PC12 cells. Increased [Ca²⁺]_i and decreased MMP may induce autophagy during reoxygenation in PC12 cells. Autophagy may be a protective effect on PC12 cells treated with different time points of reoxygenation after 2 h OGD.     

5' untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins.

5' untranslated leaders (5' UTLs) are suggested to play a crucial role in the selective translation of their eukaryotic mRNAs encoding heat shock proteins (HSP) during heat stress conditions. However, the structural features of the HSP mRNAs which cause this effect are mostly unknown. We have compiled the 5' UTLs from about 140 eukaryotic HSP mRNAs including vertebrates, invertebrates, higher and lower plants. A detailed analysis of these sequences according to length, A+T content, context of functional ATGs and presence of upstream non-functional ATGs was made. We observed that all these features were similar to the earlier studies in the literature based on data from HSP as well as non-HSP mRNAs. These observations were reconfirmed by intra-specific comparison of 5' UTLs from HSP and non-HSP genes. Similar to the translation element involved in the selective translation of mRNAs in polioviruses, a search for a short sequence motif complementary to highly conserved 18S rRNA was performed using a HSP mRNA database. The majority of the HSP mRNA sequences (77%) contained one or more small sequence motifs suggesting that they may function as internal ribosome entry sites for selective initiation of translation during heat stress.     

Oxygen availability regulates metabolism and gene expression in trout hepatocyte cultures

We studied the metabolic rate, cellular energetic state, hypoxia-inducible factor-1 (HIF-1) activation, and expression of enzymes involved in energy metabolism using rainbow trout (Oncorhynchus mykiss) hepatocytes over the oxygen range from 21 to 1 kPa. Oxygen dependence of these factors was assessed by gradually reducing oxygen supply to cells from 21 kPa to 10, 5, 2, and 1 kPa. Moreover, time course experiments for up to 20 h at oxygen tensions of 1 and 2 kPa were carried out. Reduction of oxygen from 21 kPa to 10, 5, 2, and 1 kPa decreased metabolic rate of the cells by 14, 24, 37, and 46%, respectively. This response was instantaneous and fully reversible upon reoxygenation. Cellular ATP content and the expression of all mRNAs studied decreased when oxygen was reduced from 21 to 5 and 2 kPa. The lowest ATP levels, approximately 43% of the initial value, were measured at 5 kPa of oxygen, whereas the reduction in mRNA amounts was most pronounced at 2 kPa. At 1 kPa oxygen tension, both ATP content and mRNA amounts returned to normoxic (21 kPa) levels with a concomitant activation of HIF-1, indicating reorganization of energy metabolism in adaptation of cells to low oxygen supply. These results show that oxygen has a direct regulatory effect on metabolism of trout hepatocyte cultures, supporting the view that oxygen has a profound role in metabolic regulation in cells.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.