Role In Translational Regulation Research Articles

Xtr, a plural tudor domain‐containing protein, coexists with FRGY2 both in cytoplasmic mRNP particle and germ plasm in Xenopus embryo: Its possible role in translational regulation of maternal mRNAs

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.

Biphasic activation of the mTOR pathway in the gustatory cortex is correlated with and necessary for taste learning.

Different forms of memories and synaptic plasticity require synthesis of new proteins at the time of acquisition or immediately after. We are interested in the role of translation regulation in the cortex, the brain structure assumed to store long-term memories. The mammalian target of rapamycin, mTOR (also known as FRAP and RAFT-1), is part of a key signal transduction mechanism known to regulate translation of specific subset of mRNAs and to affect learning and synaptic plasticity. We report here that novel taste learning induces two waves of mTOR activation in the gustatory cortex. Interestingly, the first wave can be identified both in synaptoneurosomal and cellular fractions, whereas the second wave is detected in the cellular fraction but not in the synaptic one. Inhibition of mTOR, specifically in the gustatory cortex, has two effects. First, biochemically, it modulates several known downstream proteins that control translation and reduces the expression of postsynaptic density-95 in vivo. Second, behaviorally, it attenuates long-term taste memory. The results suggest that the mTOR pathway in the cortex modulates both translation factor activity and protein expression, to enable normal taste memory consolidation.

Upstream sequence elements direct post-transcriptional regulation of gene expression under stress conditions in yeast

BackgroundThe control of gene expression in eukaryotic cells occurs both transcriptionally and post-transcriptionally. Although many genes are now known to be regulated at the translational level, in general, the mechanisms are poorly understood. We have previously presented polysomal gradient and array-based evidence that translational control is widespread in a significant number of genes when yeast cells are exposed to a range of stresses. Here we have re-examined these gene sets, considering the role of UTR sequences in the translational responses of these genes using recent large-scale datasets which define 5' and 3' transcriptional ends for many yeast genes. In particular, we highlight the potential role of 5' UTRs and upstream open reading frames (uORFs).ResultsWe show a highly significant enrichment in specific GO functional classes for genes that are translationally up- and down-regulated under given stresses (e.g. carbohydrate metabolism is up-regulated under amino acid starvation). Cross-referencing these data with the stress response data we show that translationally upregulated genes have longer 5' UTRs, consistent with their role in translational regulation. In the first genome-wide study of uORFs in a set of mapped 5' UTRs, we show that uORFs are rare, being statistically under-represented in UTR sequences. However, they have distinct compositional biases consistent with their putative role in translational control and are more common in genes which are apparently translationally up-regulated.ConclusionThese results demonstrate a central regulatory role for UTR sequences, and 5' UTRs in particular, highlighting the significant role of uORFs in post-transcriptional control in yeast. Yeast uORFs are more highly conserved than has been suggested, lending further weight to their significance as functional elements involved in gene regulation. It also suggests a more complex and novel mechanism of control, whereby uORFs permit genes to escape from a more general attenuation of translation under conditions of stress. However, since uORFs are relatively rare (only ~13% of yeast genes have them) there remain many unanswered questions as to how UTR elements can direct translational control of many hundreds of genes under stress.

IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA

IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.

Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in the mRNA metabolism. The absence of FMRP in neurons leads to alterations of the synaptic plasticity, probably as a result of translation regulation defects. The exact molecular mechanisms by which FMRP plays a role in translation regulation have remained elusive. The finding of an interaction between FMRP and the RNA interference silencing complex (RISC), a master of translation regulation, has suggested that both regulators could be functionally linked. We investigated here this link, and we show that FMRP exhibits little overlap both physically and functionally with the RISC machinery, excluding a direct impact of FMRP on RISC function. Our data indicate that FMRP and RISC are associated to distinct pools of mRNAs. FMRP, unlike RISC machinery, associates with the pool of mRNAs that eventually goes into stress granules upon cellular stress. Furthermore, we show that FMRP plays a positive role in this process as the lack of FMRP or a point mutant causing a severe fragile X alter stress granule formation. Our data support the proposal that FMRP plays a role in controlling the fate of mRNAs after translation arrest.

Position and Stability Are Determining Factors for Translation Repression by an RNA G-Quadruplex-Forming Sequence within the 5′ UTR of the NRAS Proto-oncogene

Nucleic acid secondary structures in the 5' untranslated regions (UTRs) of mRNAs have been shown to play a critical role in translation regulation. We recently demonstrated that a naturally occurring, conserved, and stable RNA G-quadruplex element (5'-GGGAGGGGCGGGUCUGGG-3'), located close to the 5' cap within the 5' UTR of the NRAS proto-oncogene mRNA, modulates gene expression at the translational level. Herein, we show that the translational effect of this G-quadruplex motif in NRAS 5' UTR is not uniform, but rather depends on the location of the G-quadruplex-forming sequence. The RNA G-quadruplex-forming sequence represses translation when situated relatively proximal to the 5' end, within the first 50 nt, in the 5' UTR of the NRAS proto-oncogene, whereas it has no significant effect on translation if located comparatively away from the 5' end. We have also demonstrated that the thermodynamic stability of the RNA G-quadruplex at its natural position within the NRAS 5' UTR is an important factor contributing toward its ability to repress translation.

Kill the messenger: mRNA decay and plant development

A pervasive theme in development is that dynamic changes in gene expression drive developmental progression; yet in studies of gene expression, the general RNA decay pathways have historically played second fiddle to transcription. However, recent advances in this field have revealed a surprising degree of mRNA specificity for particular branches of these RNA decay pathways. General cytoplasmic mRNA decay typically initiates with deadenylation, following which the deadenylated mRNA can continue decay from the 3'-end through the action of the exosome, or it can undergo 5'-to-3' decay. Functional characterization of exosome subunits using inducible knock-outs uncovered a surprising complexity of molecular phenotypes and RNA substrates. Decay in the 5'-to-3' direction requires decapping, which is carried out by the decapping complex in Processing bodies (PBs). Recent analyses of decapping mutants have also revealed substrate specificity and roles in translational regulation. In addition, recent studies of specialized pathways such as nonsense-mediated decay and silencing reveal interactions with the general RNA decay pathways.

A Small Molecule Inhibitor of Isoprenylcysteine Carboxymethyltransferase Induces Autophagic Cell Death in PC3 Prostate Cancer Cells

A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336-4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G(1) phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G(1) cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy.

Roles of Helicobacter pylori BabA in gastroduodenal pathogenesis

Interactions between BabA and Lewis b (Le(b)) related antigens are the best characterized adhesin-receptor interactions in Helicobacter pylori (H pylori). Several mechanisms for the regulation of BabA expression are predicted, including at both transcriptional and translational levels. The formation of chimeric proteins (babA/B or babB/A chimeras) seems to play an especially important role in translational regulation. Chimeric BabB/A protein had the potential to bind Le(b); however, protein production was subject to phase variation through slipped strand mispairing. The babA gene was cloned initially from strain CCUG17875, which contains a silent babA1 gene and an expressed babA2 gene. The sequence of these two genes differs only by the presence of a 10 bp deletion in the signal peptide sequence of babA1 that eliminates its translational initiation codon. However, the babA1 type deletion was found only in strain CCUG17875. A few studies evaluated BabA status by immunoblot and confirmed that BabA-positive status in Western strains was closely associated with severe clinical outcomes. BabA-positive status also was associated with the presence of other virulence factors (e.g. cagA-positive status and vacA s1 genotype). A small class of strains produced low levels of the BabA protein and lacked Le(b) binding activity. These were more likely to be associated with increased mucosal inflammation and severe clinical outcomes than BabA-positive strains that exhibited Le(b) binding activity. The underlying mechanism is unclear, and further studies will be necessary to investigate how the complex BabA-receptor network is functionally coordinated during the interaction of H pylori with the gastric mucosa.

Drosophila NAT1, a homolog of the vertebrate translational regulator NAT1/DAP5/p97, is required for embryonic germband extension and metamorphosis

Translational regulation has been to shown to play major roles in the patterning of the early Drosophila embryo. The eIF4G family member NAT1/p97/DAP5 has been identified as a novel translational repressor. To genetically dissect the in vivo function of this unconventional eIF4G-related translational regulator, Drosophila NAT1 (dNAT1) mutants were isolated using a reverse-genetics approach. Four transposon insertion mutants and a deletion mutant affecting the dNAT1 locus were analyzed. Genetic complementation tests and germline rescue using a 12 kb dNAT1 genomic DNA fragment revealed these to be loss-of-function mutants. One P-element insertion line, dNAT1(GS1.), shows severe embryonic lethality and abnormal germband extension. Abnormalities at metamorphosis were also found, including defective head eversion and salivary gland degeneration in the hypomorphic allele dNAT(ex1). A phenotypic analysis of dNAT1 mutants suggests that dNAT protein plays a specific rather than general role in translational regulation.

Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

BackgroundThe translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis.ResultsWe have used comparative genomics to identify novel uORFs in yeast with a high likelihood of having a translational regulatory role. We examined uORFs, previously shown to play a role in regulation of translation in Saccharomyces cerevisiae, for evolutionary conservation within seven Saccharomyces species. Inspection of the set of conserved uORFs yielded the following three characteristics useful for discrimination of functional from spurious uORFs: a length between 4 and 6 codons, a distance from the start of the main ORF between 50 and 150 nucleotides, and finally a lack of overlap with, and clear separation from, neighbouring uORFs. These derived rules are inherently associated with uORFs with properties similar to the GCN4 locus, and may not detect most uORFs of other types. uORFs with high scores based on these rules showed a much higher evolutionary conservation than randomly selected uORFs. In a genome-wide scan in S. cerevisiae, we found 34 conserved uORFs from 32 genes that we predict to be functional; subsequent analysis showed the majority of these to be located within transcripts. A total of 252 genes were found containing conserved uORFs with properties indicative of a functional role; all but 7 are novel. Functional content analysis of this set identified an overrepresentation of genes involved in transcriptional control and development.ConclusionEvolutionary conservation of uORFs in yeasts can be traced up to 100 million years of separation. The conserved uORFs have certain characteristics with respect to length, distance from each other and from the main start codon, and folding energy of the sequence. These newly found characteristics can be used to facilitate detection of other conserved uORFs.

Eukaryotic translation initiation factor 4E (eIF4E) expression in the brain tissue is induced by infusion of nerve growth factor into the mouse cisterna magnum: an in vivo study

In many cell types translation can be regulated by an expression of the translation initiation factor. Eukaryotic translation initiation factor eIF4E, which binds to the 5' cap structure of mRNA, plays an important role in translation regulation and it has been suggested that it is implicated in increased protein synthesis promoted by growth factors. In this study the effects of nerve growth factor (NGF) infusion into the cerebrospinal fluid (CSF) on eIF4E expression and phosphorylation in mouse brain tissue have been investigated. We investigated NGF as it is one of the most important growth factors and it is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation. eIF4E level is also increased in response to infusion of NGF into the CSF. The present study shows that eIF4E is phosphorylated in the brain tissues treated with NGF. It is concluded that NGF regulates protein synthesis in the nervous tissue by enhancing expression and phosphorylation of eIF4E.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

The GI‐UEV Domain, a Catalytically Inactive Ubiquitin‐Conjugating Enzyme Variant With a Role in Translational Regulation

AbstractUbiquitin‐conjugating enzymes (UBCs) form the second step in the enzyme cascade required for protein ubiquitylation. Most eukaryotic genomes contain a multitude of different catalytically active UBCs. In addition, several proteins contain homology domains related to UBCs that have lost their catalytic activity and are referred to as “ubiquitin‐conjugating enzyme variants” or UEV‐domains. A common property of those domains is a role in ubiquitin binding and recognition. We report here on a novel class of more distantly related UEV domains, which forms a superset of the previously described GI‐homology region found in Gcn2 and IMPACT proteins. In the Gcn2 and IMPACT protein families, the GI‐UEV domain binds to proteins of the Gcn1 family and thus regulates translation levels via eIF2α phosphorylation. Bioinformatical analysis shows that GI‐UEV domains occur in a large number of proteins, many of them without an established role in translational regulation. Residue conservation and domain context predict that GI‐UEV domains might also have a role in the ubiquitin/proteasome system, suggesting a possible cross‐talk between ubiquitylation and translational regulation.

Ataxin-2 and its Drosophila homolog, ATX2, physically assemble with polyribosomes

Mutations resulting in the expansion of a polyglutamine tract in the protein ataxin-2 give rise to the neurodegenerative disorders spinocerebellar ataxia type 2 and Parkinson's disease. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration are unknown. Here, we demonstrate that ataxin-2 and its Drosophila homolog, ATX2, assemble with polyribosomes and poly(A)-binding protein (PABP), a key regulator of mRNA translation. The assembly of ATX2 with polyribosomes is mediated independently by two distinct evolutionarily conserved regions of ATX2: an N-terminal Lsm/Lsm-associated domain (LsmAD), found in proteins that function in nuclear RNA processing and mRNA decay, and a PAM2 motif, found in proteins that interact physically with PABP. We further show that the PAM2 motif mediates a physical interaction of ATX2 with PABP in addition to promoting ATX2 assembly with polyribosomes. Our results suggest a model in which ATX2 binds mRNA directly through its Lsm/LsmAD domain and indirectly via binding PABP that is itself directly bound to mRNA. These findings, coupled with work on other ataxin-2 family members, suggest that ATX2 plays a direct role in translational regulation. Our results raise the possibility that polyglutamine expansions within ataxin-2 cause neurodegeneration by interfering with the translational regulation of particular mRNAs.

A novel class of small RNAs in mouse spermatogenic cells

Small noncoding RNAs, including small interfering RNAs (siRNAs) and micro RNAs (miRNAs) of approximately 21 nucleotides (nt) in length, have emerged as potent regulators of gene expression at both transcriptional and post-transcriptional levels in diverse organisms. Here we report the identification of a novel class of small RNAs in the mouse male germline termed piwi-interacting RNAs (piRNAs). piRNAs are approximately 30 nt in length. They are expressed during spermatogenesis, mostly in spermatids. piRNAs are associated with MIWI, a spermatogenesis-specific PIWI subfamily member of the Argonaute protein family, and depend on MIWI for their biogenesis and/or stability. Furthermore, a subpopulation of piRNAs are associated with polysomes, suggesting their potential role in translational regulation.

Interaction between RAX and PKR Modulates the Effect of Ethanol on Protein Synthesis and Survival of Neurons

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.

The Eukaryotic mRNA Cycle: Movement of yeast mRNAs between polysomes and P‐bodies and its role in translational regulation.

The Eukaryotic mRNA Cycle: Movement of yeast mRNAs between polysomes and P‐bodies and its role in translational regulation.

Mammalian Target of Rapamycin Is Required for Thrombopoietin-Induced Proliferation of Megakaryocyte Progenitors

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34+, and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival.

UAUG and uORFs in human and rodent 5′untranslated mRNAs

The control of translation is a fundamental mechanism in the regulation of gene expression. Among the cis-acting elements that play a role in translation regulation are upstream open reading frames (uORFs) and upstream AUG (uAUGs) located in the 5′UTR of mRNAs. We present here a genome-wide analysis of uAUGs and uORFs in a curated set of human and rodent mRNAs. Our study shows that the occurrence of uAUGs is suppressed more strongly than that of uORFs and that in-frame uAUGs are more strongly suppressed than out-of-frame uAUGs. A very similar pattern of uAUG/uORF frequency was also observed in mouse mRNAs. The analysis of orthologous 5′UTR sequences revealed a remarkable degree of evolutionary conservation only of those uORFs which acquired some functional activity. Our data suggest that besides leaky scanning and reinitiation, which likely occur with variable and gene-specific efficiency, the ribosome-shunt mechanism, eventually coupled to reinitiation after uORF translation, may be a widespread mode of translation regulation in eukaryotes.