Orna Elroy-Stein

Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription.

Vascular Endothelial Growth Factor (VEGF) is a very potent angiogenic agent that has a central role in normal physiological angiogenesis as well as in tumor angiogenesis. VEGF expression is induced by hypoxia and hypoglycemia, and thus was suggested to promote neovascularization during tumor outgrowth. Yet, the molecular mechanism that governs VEGF expression is not fully characterized. VEGF induction is attributed in part to increased levels of transcription and RNA stability. Previously, we demonstrated that the 5′ Untranslated Region (5′ UTR) of VEGF has an important regulatory role in its expression. VEGF has an exceptionally long 5′ UTR (1038 bp) which is highly rich in G+C nucleotides. This suggests that secondary structures in the 5′ UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms, as demonstrated for other growth factors. In this communication, we provide evidence that a computer predicted Internal Ribosome Entry Site (IRES) structure is biologically active and is located at the 3′ end of the UTR. In addition, the results demonstrate that an alternative transcriptional initiation site for VEGF exists in the 5′ UTR of VEGF. This alternative initiation site is 633 bp downstream of the main transcription start site and the resulting 5′ UTR includes mainly the IRES structure. Therefore, our results suggest that VEGF is subjected to regulation at either translational level through a mechanism of ribosome internal initiation and/or transcriptional level through alternative initiation.

Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins Cytokine receptor-like molecules

Phosphorylation on tyrosine residues is a key step in signal transduction pathways mediated by membrane proteins. Although it is known that human breast cancer tissue expresses at least 2 MUC1 type 1 membrane proteins (a polymorphic high molecular weight MUC1 glycoprotein that contains a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein that is not polymorphic and is lacking this repeat array) their function in the development of human breast cancer has remained elusive. Here it is shown that these MUC1 proteins are extensively phosphorylated, that phosphorylation occurs primarily on tyrosine residues and that following phosphorylation the MUC1 proteins may potentially interact with SH2 domain‐containing proteins and thereby initiate a signal transduction cascade. As with cytokine receptors, the MUC1 proteins do not harbor intrinsic tyrosine kinase activity yet are tyrosine phosphorylated and the MUC1/Y protein participates in a cell surface heteromeric complex whose formation is mediated by two cytoplasmically located MUC1 cysteine residues. Furthermore, the MUC1/Y protein demonstrates sequence similarity with sequences present in cytokine receptors that are known to be involved in ligand binding. Our results demonstrate that the two MUC1 isoforms are both likely to function in signal transduction pathways and to be intimately linked to the oncogenetic process and suggest that the MUC1/Y protein may act in a similar fashion to cytokine receptors.

Transcription-Coupled Translation Control of AML1/RUNX1 Is Mediated by Cap- and Internal Ribosome Entry Site-Dependent Mechanisms

ABSTRACT AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5′ untranslated regions (5′UTRs) (D-UTR and P-UTR). Here we show that these 5′UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

Differentiation-Induced Internal Translation of c-sis mRNA: Analysis of the cis Elements and Their Differentiation-Linked Binding to the hnRNP C Protein

ABSTRACT In previous reports we showed that the long 5′ untranslated region (5′ UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiation-activated internal ribosomal entry site (D-IRES). Here we show that the 5′ UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5′ UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5′ UTR. Even though the region responsible for IRES activation is smaller, the full-length 5′ UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5′ UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiation-sensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5′ UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulatedtrans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis.

Ribosomal Slowdown Mediates Translational Arrest during Cellular Division

ABSTRACT Global mRNA translation is transiently inhibited during cellular division. We demonstrate that mitotic cells contain heavy polysomes, but these are significantly less translationally active than polysomes in cycling cells. Several observations indicate that mitotic translational attenuation occurs during the elongation stage: (i) in cycling nonsynchronized cultures, only mitotic cells fail to assemble stress granules when treated with agents that inhibit translational initiation; (ii) mitotic cells contain fewer free 80S complexes, which are less sensitive to high salt disassembly; (iii) mitotic polysomes are more resistant to enforced disassembly using puromycin; and (iv) ribosome transit time increases during mitosis. Elongation slowdown guarantees that polysomes are retained even if initiation is inhibited at the same time. Stalling translating ribosomes during mitosis may protect mRNAs and allow rapid resumption of translation immediately upon entry into the G1 phase.

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.

Species-specific microRNA roles elucidated following astrocyte activation

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in regulation of gene expression by binding to target genes. Many miRNAs were associated with the function of the central nervous system (CNS) in health and disease. Astrocytes are the CNS most abundant glia cells, providing support by maintaining homeostasis and by regulating neuronal signaling, survival and synaptic plasticity. Astrocytes play a key role in repair of brain insults, as part of local immune reactivity triggered by inflammatory or pathological conditions. Thus, astrocyte activation, or astrogliosis, is an important outcome of the innate immune response, which can be elicited by endotoxins such as lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-γ). The involvement of miRNAs in inflammation and stress led us to hypothesize that astrogliosis is mediated by miRNA function. In this study, we compared the miRNA regulatory layer expressed in primary cultured astrocyte derived from rodents (mice) and primates (marmosets) brains upon exposure to LPS and IFN-γ. We identified subsets of differentially expressed miRNAs some of which are shared with other immunological related systems while others, surprisingly, are mouse and rat specific. Of interest, these specific miRNAs regulate genes involved in the tumor necrosis factor-alpha (TNF-α) signaling pathway, indicating a miRNA-based species-specific regulation. Our data suggests that miRNA function is more significant in the mechanisms governing astrocyte activation in rodents compared to primates.

Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.

A mouse model for eukaryotic translation initiation factor 2B-leucodystrophy reveals abnormal development of brain white matter

Eukaryotic translation initiation factor 2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. Mutations in any of its five subunits lead to leucoencephalopathy with vanishing white matter, an inherited chronic-progressive fatal brain disease with unknown aetiology, which is among the most prevalent childhood white matter disorders. We generated the first animal model for the disease by introducing a point mutation into the mouse Eif2b5 gene locus, leading to R132H replacement corresponding to the clinically significant human R136H mutation in the catalytic subunit. In contrast to human patients, mice homozygous for the mutant Eif2b5 allele (Eif2b5(R132H/R132H) mice) enable multiple analyses under a defined genetic background during the pre-symptomatic stages and during recovery from a defined brain insult. Time-course magnetic resonance imaging revealed for the first time the delayed development of the brain white matter due to the mutation. Electron microscopy demonstrated a higher proportion of small-calibre nerve fibres. Immunohistochemistry detected an abnormal abundance of oligodendrocytes and astrocytes in the brain of younger animals, as well as an abnormal level of major myelin proteins. Most importantly, mutant mice failed to recover from cuprizone-induced demyelination, reflecting an increased sensitivity to brain insults. The anomalous development of white matter in Eif2b5(R132H/R132H) mice underscores the importance of tight translational control to normal myelin formation and maintenance.

Mitotic Modulation of Translation Elongation Factor 1 Leads to Hindered tRNA Delivery to Ribosomes

Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.