Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model

Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5' end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.     

The AU-rich sequences in the 3'' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids

Different vectors were constructed that expressed the human interferon-beta (IFN-beta) mRNA constitutively and contained various deletions in the 3' untranslated region (UTR). AU-rich sequences in the 3' UTR were specifically deleted in two vectors. Cell lines secreting human IFN-beta were established by transfecting murine L929 cells with the vectors. These cells showed similar levels of IFN-beta mRNA and secreted comparable amounts of IFN-beta, indicating that the deletion of AU-rich sequences had no effect on the stability and little effect on the efficiency of translation of this mRNA. The synthetic glucocorticoid dexamethasone was previously shown to increase the turnover of IFN-beta mRNA. This activity of dexamethasone was clearly observed only in cells expressing IFN-beta mRNA with AU-rich sequences in the 3' UTR. The increased turnover of this mRNA occurred in the presence of cycloheximide; therefore, it did not require synthesis of new proteins. These findings suggest that glucocorticoids may activate a ribonuclease that degrades mRNAs containing AU-rich sequences in the 3' UTR.     

The poly(A)-binding protein partner Paip2a controls translation during late spermiogenesis in mice

Translational control plays a key role in late spermiogenesis. A number of mRNAs encoding proteins required for late spermiogenesis are expressed in early spermatids but are stored as translationally inactive messenger ribonucleoprotein particles (mRNPs). The translation of these mRNAs is associated with shortening of their poly(A) tail in late spermiogenesis. Poly(A)-binding protein (Pabp) plays an important role in mRNA stabilization and translation. Three Pabp-interacting proteins, Paip1, Paip2a, and Paip2b, have been described. Paip2a is expressed in late spermatids. To investigate the role of Paip2 in spermiogenesis, we generated mice with knockout of either Paip2a or Paip2b and double-KO (DKO) mice lacking both Paip2a and Paip2b. Paip2a-KO and Paip2a/Paip2b-DKO mice exhibited male infertility. Translation of several mRNAs encoding proteins essential to male germ cell development was inhibited in late spermiogenesis in Paip2a/Paip2b-DKO mice, resulting in defective elongated spermatids. Inhibition of translation in Paip2a/Paip2b-DKO mice was caused by aberrant increased expression of Pabp, which impaired the interaction between eukaryotic initiation factor 4E (eIF4E) and the cap structure at the 5′ end of the mRNA. We therefore propose a model whereby efficient mRNA translation in late spermiogenesis occurs at an optimal concentration of Pabp, a condition not fulfilled in Paip2a/Paip2b-DKO mice.     

A mutation in the 5' untranslated region of the BRCA1 gene in sporadic breast cancer causes downregulation of translation efficiency

We screened 117 breast tumour samples in Chinese females for mutations in the breast cancer 1 (BRCA1) gene and identified a novel mutation in the 5' untranslated region (5' UTR) in two patients with grade III infiltrating ductal breast carcinoma. We examined whether this 5' UTR mutation affected the translational efficiency of BRCA1 protein. A vector was constructed containing the mutated 5' UTR up-stream of luciferase and we compared its translational efficiency with a wild-type 5' UTR. The expression of BRCA1 protein in breast tumour samples was evaluated using immunohistochemistry. The mutated 5' UTR of BRCA1 resulted in less luciferase activity compared with the wild-type 5' UTR, while there were no significant differences in luciferase mRNA levels. BRCA1 protein was much less expressed in breast tumour tissue from patients with the 5' UTR mutation than in samples from patients without the mutation. Our results show that a mutation in the 5' UTR of the BRCA1 gene downregulates translational efficiency of the protein.     

The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice

In both type 1 and type 2 diabetes, pancreatic islet dysfunction results in part from cytokine-mediated inflammation. The ubiquitous eukaryotic translation initiation factor 5A (eIF5A), which is the only protein to contain the amino acid hypusine, contributes to the production of proinflammatory cytokines. We therefore investigated whether eIF5A participates in the inflammatory cascade leading to islet dysfunction during the development of diabetes. As described herein, we found that eIF5A regulates iNOS levels and that eIF5A depletion as well as the inhibition of hypusination protects against glucose intolerance in inflammatory mouse models of diabetes. We observed that following knockdown of eIF5A expression, mice were resistant to β cell loss and the development of hyperglycemia in the low-dose streptozotocin model of diabetes. The depletion of eIF5A led to impaired translation of iNOS-encoding mRNA within the islet. A role for the hypusine residue of eIF5A in islet inflammatory responses was suggested by the observation that inhibition of hypusine synthesis reduced translation of iNOS-encoding mRNA in rodent β cells and human islets and protected mice against the development of glucose intolerance the low-dose streptozotocin model of diabetes. Further analysis revealed that hypusine is required in part for nuclear export of iNOS-encoding mRNA, a process that involved the export protein exportin1. These observations identify the hypusine modification of eIF5A as a potential therapeutic target for preserving islet function under inflammatory conditions.     

Leucine acutely reverses burn-induced alterations in translation initiation in heart

Myocardial dysfunction is a common manifestation of thermal injury, the etiology of which appears to be multifactorial. We have previously demonstrated that burn injury impairs cardiac protein synthesis at the level of translation initiation. The purpose of the present study was to determine whether oral administration of leucine, which is known to stimulate translation initiation in skeletal muscle, can ameliorate burn-induced changes in signal transduction pathways known to regulate protein synthesis in cardiac muscle. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied in the fasted condition 24 h later; appropriate time-matched nonburned control rats were also included. Separate groups of control and burn rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined using immunoprecipitation and immunoblotting techniques. Hearts from burned rats demonstrated a redistribution of eIF4E as evidenced by the increased binding of the translational repressor 4E-BP1 with eIF4E, a decreased amount of eIF4E bound with eIF4G, and a decreased amount of the hyperphosphorylated gamma-isoform of 4E-BP1. Furthermore, constitutive phosphorylation of mTOR, the ribosomal protein S6, and eIF4G was also decreased in hearts from burned rats. In control rats, leucine failed to alter eIF4E distribution but did increase the phosphorylation of S6K1 and S6. However, in hearts from burn rats, leucine acutely reversed the alterations in eIF4E distribution as well as the changes in S6, eIF4G, and mTOR phosphorylation. These data suggest that oral administration of leucine can acutely reverse multiple defects in cardiac translation initiation produced by thermal injury.     

FXTAS: a bad RNA and a hope for a cure

Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a newly identified neurodegenerative disorder due to intermediate expansion of trinucleotide CGG repeats (55 - 200 repeats) in the 5' untranslated region (UTR) of the Fragile X mental retardation 1 (FMR1) gene. FXTAS is now considered to be one of the most common inherited neurodegenerative disorders in males. Objective: To examine the future of potential therapies for this late-onset disease. Methods: Examination of relevent literature. Results/conclusions: Accumulating evidence indicates that overproduced riboCGG repeats in the 5' UTR of FMR1 mRNA are toxic. Recently, proteins that bind specifically to rCGG repeats were identified. Progress in understanding the molecular pathogenesis of FXTAS, plus the availability of different animal models are discussed.     

Diminished ERK 1/2 and p38 MAPK phosphorylation in skeletal muscle during sepsis

Sepsis induces weight loss and the loss of skeletal muscle proteins, in part through an inhibition of protein synthesis secondary to an inhibition of the key steps controlling mRNA translation in skeletal muscle. We have previously shown that sepsis decreases the phosphorylation of eIF4E. The present study examines the phosphorylation of Erk 1/2 MAPK and p38 MAPK in skeletal muscle of rats with a chronic (5-day) intra-abdominal septic abscess. Mnk1 catalyzes the phosphorylation of eIF4E, and Mnk1 is activated by phosphorylation via Erk1/2 MAPK and p38 MAPK. Sepsis resulted in a significant decrease in the steady-state phosphorylation of Erk 1/2 and p38 MAPKs compared with sterile inflammation. To examine the mediators responsible for decreased phosphorylation of Erk 1/2 and p38 MAPKs, rats were treated with TNF binding protein (TNFbp) or infused for 24 h with TNF. Treatment of septic rats with TNFbp resulted in an increase in the phosphorylation of both Erk 1/2 and p38 MAPKs in skeletal muscle. This was associated with enhanced phosphorylation of eIF4E. In contrast, constant intravenous infusion of TNF-alpha for 24 h resulted in a complete inhibition of p38 MAPK phosphorylation while Erk 1/2 MAPK phosphorylation was increased. The net effect was a modest increase in eIF4E phosphorylation. The results suggest altered regulation of Erk 1/2 and p38 MAPK signal translation pathways by endogenously produced TNF, or some compound dependent on TNF may modulate, in part, the phosphorylation state of eIF4E in skeletal muscle during sepsis.     

Translational profiles of alpha 1-, alpha 2-, and beta-globin messenger ribonucleic acids in human reticulocytes.

In human reticulocytes, the critical balancing of alpha- and beta-globin synthesis may be controlled in part by differential translation of the three major adult globin messenger RNAs (mRNAs), alpha 1, alpha 2, and beta. In this study, we determined, as a parameter of translational efficiency, the relative ribosome loading of these three mRNAs. Using oligonucleotide probes specific for the alpha 1- and alpha 2-globin mRNAs, we find that these two mRNAs have identical translational profiles. Their distribution contrasts with that of beta-globin mRNA, which is present on heavier polyribosomes and is less prevalent in pre-80S messenger ribonucleoprotein fractions. The relative distribution of alpha- vs. beta-globin mRNA is consistent with more efficient beta-globin translation. In contrast, the parallel distributions of alpha 1- and alpha 2-globin mRNAs suggests they are translated with equal efficiencies. Considering the relative concentrations of the two alpha-globin mRNAs in normal reticulocytes, this result predicts a dominant role for the alpha 2-globin locus in human alpha-globin expression.     

Ethanol destabilizes liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase, mRNA by depleting a 3'-untranslated region-specific binding protein

Asialoconjugates are viable biomarkers for alcohol abuse. We previously showed that chronic ethanol feeding down-regulated liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase (ST6Gal l) mRNA by destabilizing it. Since RNA-binding proteins are known to stabilize many eukaryotic mRNAs by interacting with the 3'-untranslated region (UTR), we have delineated the possible mechanism by which ethanol destabilizes ST6Gal l mRNA. Using (32)P-labeled RNA probes generated from a 2.7-kb 3'-UTR of ST6Gal l mRNA, we identified a liver cytosolic 41-kDa specific binding protein that interacts with its 3'-UTR domain and protects it from degradation in normal rat liver but disappears after chronic ethanol treatment. Mapping of the binding region revealed that four RNA probes of 80-base pair (bp) length spanning the 304 bp of the 3'-UTR of ST6Gal l mRNA showed equal binding intensity. The corresponding cDNA sequences for the four 80-bp RNA probes share the 13-bp consensus sequence. Mutagenesis analysis identified that four nucleotides, AG and TC, among the consensus sequences were critical for the RNA-protein interaction. Therefore, 5'-CAGCCTCCTCCCT-3' serves as a cis-element critically involved in this interaction. The RNA-protein complex formation progressively decreased with increasing dietary ethanol, resulting in its virtual disappearance with 36% of the dietary calories as ethanol. Concomitantly, the same ethanol diet decreased sialic acid index of plasma apolipoprotein J by 45% (p < 0.05). Thus, depletion of a binding protein that specifically interacts with its 3'-UTR region of ST6Gal l mRNA may account for its destabilization and consequent appearance of asialoconjugates as alcohol biomarkers.