Stress-Induced Translational Control in Potato Tubers May Be Mediated by Polysome-Associated Proteins

Potato tubers exhibit distinct responses to wounding and hypoxia that include selective translation of stress-induced mRNAs. Newly synthesized wound-response mRNAs are bound to polysomes, whereas preexisting mRNAs are displaced and degraded. mRNAs that are induced and translated during hypoxic conditions are bound to ribosomes as expected. However, preexisting wound-response mRNAs whose translation is inhibited during hypoxia remain bound to polysomes, indicating that there are at least two distinct mechanisms by which translation is regulated in response to stress conditions. A 32-kD phosphoprotein is associated with polyribosomes from wounded tubers. This protein remains polysome bound as long as wound-response mRNAs are present, even during hypoxia when these mRNAs are no longer translated. However, association of the 32-kD protein with polysomes is not elicited by hypoxic stress alone. The kinase that phosphorylates this protein is active only for the first 24 hr after wounding and is not active during periods of hypoxia. This protein may mediate recognition of the wound-response mRNAs by ribosomes.     

Tissue-specific expression of major urinary protein (MUP) genes in mice: characterization of MUP mRNAs by restriction mapping of cDNA and by in vitro translation.

The major urinary proteins (MUPs) in mice are coded for by a gene family which consists of ca. 30 members. The number of MUP genes that are expressed is not known. Previous studies have shown that MUP mRNAs are present in several secretory tissues in addition to the liver, in which they were originally identified. In this paper we show, through restriction analysis of MUP cDNAs, that distinct sets of MUP mRNAs are synthesized in each of the tissues studied and that these mRNAs are most likely coded for by different genes. As is shown, MUP mRNAs of different tissues are related to an extent that precludes the use of gene-specific probes in differentiating among them. The regions of homology also include the 3' untranslated regions of MUP mRNAs. The question of differential expression was thus investigated by searching for restriction polymorphisms in MUP mRNAs. We demonstrate that subtle differences in the sequences of even scarce mRNAs can be recognized by this particular approach. In addition, it is shown that MUP mRNAs of different tissues code for different, nonoverlapping sets of polypeptides, as determined by gel electrophoresis of in vitro-translated precursors to MUPs. The relevance of these results to models of evolution of tissue-specific regulation in a multigene family is discussed.     

Translational discrimination of ribosomal protein mRNAs in the early Drosophila embryo

Most Drosophila mRNAs are actively translated in the early embryo, with the exception of the poorly translated ribosomal protein (r-protein) mRNAs. Two possible mechanisms for this translational discrimination were tested: (1) Translation of r-protein mRNAs is discriminated against by the limited activity of translational initiation factors in the early embryo and (2) translation of r-protein mRNAs is repressed by trans-acting factors that reversibly bind these mRNAs. Exogenously provided initiation factors promoted partial recruitment of r-protein mRNAs into polysomes, suggesting that modulation of initiation factor activity may play a role in the translational discrimination of r-protein mRNAs during embryogenesis. No evidence for involvement of reversibly binding trans-acting factors was obtained, although there are limitations in the interpretation of the latter experiments.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Myelin-Associated Oligodendrocytic Basic Protein mRNAs Reside at Different Subcellular Locations

The mRNAs for two myelin proteins, myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP)-81A, are uniquely located at sites where myelin sheaths are assembled. Here, we use subcellular fractionation to show that four MOBP mRNAs, like MBP mRNA, are located at sites of myelin sheath assembly, and that three other MOBP mRNAs are located in oligodendrocyte soma. The MOBP-81 protein is found in myelin and in another subcellular fraction, whereas other myelin proteins, including MBP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin-associated glycoprotein, are largely restricted to myelin. Different MBP mRNAs are generated by alternative splicing. All of them contain an RNA transport sequence (RTS) that directs them to sites in oligodendrocytes, where myelin sheaths are assembled. Consequently, all are enriched in myelin. After fractionation, four MOBP mRNAs, MOBP-71, MOBP-81A, MOBP-99, and MOBP-169 (identified in this study), are enriched in myelin. These mRNAs contain a common exon, exon 8b, which has a nucleotide sequence that is similar to MBP mRNA RTS. This sequence likely directs these mRNAs to sites of myelin sheath assembly. Three other MOBP mRNAs, MOBP-69, MOBP-81B, and MOBP-170, lack this exon. Their subcellular distribution indicates that they are largely retained in oligodendrocyte soma. We conclude that the distribution of MOBPs in oligodendrocytes is strongly influenced by alternative splicing of the corresponding mRNAs.     

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.     

Lithium can relieve translational repression of TOP mRNAs elicited by various blocks along the cell cycle in a glycogen synthase kinase-3- and S6-kinase-independent manner

TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5'-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G(0), G(1)/S, or G(2)/M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.     

Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) -- bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.     

Messenger RNA degradation in Saccharomyces cerevisiae

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.     

Growth Hormone-Regulated mRNAs and miRNAs in Chicken Hepatocytes

Growth hormone (GH) is a key regulatory factor in animal growth, development and metabolism. Based on the expression level of the GH receptor, the chicken liver is a major target organ of GH, but the biological effects of GH on the chicken liver are not fully understood. In this work we identified mRNAs and miRNAs that are regulated by GH in primary hepatocytes from female chickens through RNA-seq, and analyzed the functional relevance of these mRNAs and miRNAs through GO enrichment analysis and miRNA target prediction. A total of 164 mRNAs were found to be differentially expressed between GH-treated and control chicken hepatocytes, of which 112 were up-regulated and 52 were down-regulated by GH. A total of 225 chicken miRNAs were identified by the RNA-Seq analysis. Among these miRNAs 16 were up-regulated and 1 miRNA was down-regulated by GH. The GH-regulated mRNAs were mainly involved in growth and metabolism. Most of the GH-upregulated or GH-downregulated miRNAs were predicted to target the GH-downregulated or GH-upregulated mRNAs, respectively, involved in lipid metabolism. This study reveals that GH regulates the expression of many mRNAs involved in metabolism in female chicken hepatocytes, which suggests that GH plays an important role in regulating liver metabolism in female chickens. The results of this study also support the hypothesis that GH regulates lipid metabolism in chicken liver in part by regulating the expression of miRNAs that target the mRNAs involved in lipid metabolism.     

Glucocorticoids selectively inhibit translation of ribosomal protein mRNAs in P1798 lymphosarcoma cells.

When P1798 murine lymphosarcoma cells are exposed to 10(-7) M dexamethasone, there is a dramatic inhibition of rRNA synthesis, which is completely reversible when the hormone is withdrawn. In the present experiments we examined whether dexamethasone treatment causes any alteration in the accumulation or utilization of mRNAs that encode ribosomal proteins (rp mRNAs). No effect on the accumulation of six different rp mRNAs was detected. However, the translation of five of six rp mRNAs was selectively inhibited in the presence of the hormone, as judged by a substantial decrease in ribosomal loading. Normal translation of rp mRNA was resumed within a few hours after hormone withdrawal. In untreated or fully recovered cells, the distribution of rp mRNAs between polyribosomes and free ribonucleoprotein is distinctly bimodal, suggesting that rp mRNAs are subject to a particular form of translational control in which they are either translationally inactive or fully loaded with ribosomes. A possible relationship between this mode of translational control and the selective suppression of rp mRNA translation by glucocorticoids is discussed.     

Genome-wide analysis of GLD-1-mediated mRNA regulation suggests a role in mRNA storage

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are "stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1-dependent repression prevents precocious translation of OET-promoting mRNAs. Secondly, GLD-1- and CGH-1-dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised.     

Diverse aberrancies target yeast mRNAs to cytoplasmic mRNA surveillance pathways

Eukaryotic gene expression is a complex, multistep process that needs to be executed with high fidelity and two general methods help achieve the overall accuracy of this process. Maximizing accuracy in each step in gene expression increases the fraction of correct mRNAs made. Fidelity is further improved by mRNA surveillance mechanisms that degrade incorrect or aberrant mRNAs that are made when a step is not perfectly executed. Here, we review how cytoplasmic mRNA surveillance mechanisms selectively recognize and degrade a surprisingly wide variety of aberrant mRNAs that are exported from the nucleus into the cytoplasm.     

Reassessment of the Role of TSC,mTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP mRNAs

TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.     

Formation and stability of cytoplasmic mRNAs during myoblast differentiation: Pulse-chase and density labeling analyses

Stabilities and rates of formation of cytoplasmic mRNAs have been measured quantitatively in cultures of embryonic quail breast myoblasts undergoing differentiation to form muscle fibers. Uridine pulse-chase studies show that dividing myoblasts and differentiated fibers form both short- and long-lived mRNAs. Short-lived mRNAs in myoblasts and fibers have similar half-lives of 2–4 hr, however, long-lived mRNAs have a half-life of 60–100 hr in myoblasts and only 20 hr in fibers. When myoblasts fuse, the formation of long-lived cytoplasmic mRNAs increases at least twofold, and this increased formation together with the cessation of myoblast cell division at fusion is sufficient to account for the four- to fivefold accumulation of long-lived mRNA observed in fibers. These long-lived mRNAs were identified by density labeling cultures with ¹⁵N, ¹³C-nucleotides, chasing with light nucleosides, and then translating the density labeled mRNAs in wheat germ extracts. These experiments show that the contractile protein mRNAs, as well as 60–70 other muscle mRNAs are actively synthesized by muscle fibers and that all of the specific mRNAs detected have half-lives clustering around 20 hr.