Cap-Independent Translation in Hematological Malignancies.

Hematological malignancies are a heterogeneous group of diseases deriving from blood cells progenitors. Although many genes involved in blood cancers contain internal ribosome entry sites (IRESes), there has been only few studies focusing on the role of cap-independent translation in leukemia and lymphomas. Expression of IRES trans-acting factors can also be altered, and interestingly, BCL-ABL1 fusion protein expressed from "Philadelphia" chromosome, found in some types of leukemia, regulates several of them. A mechanism involving c-Myc IRES and cap-independent translation and leading to resistance to chemotherapy in multiple myeloma emphasize the contribution of cap-independent translation in blood cancers and the need for more work to be done to clarify the roles of known IRESes in pathology and response to chemotherapeutics

Cap-Independent Translation in Hematological Malignancies.

Hematological malignancies are a heterogeneous group of diseases deriving from blood cells progenitors. Although many genes involved in blood cancers contain internal ribosome entry sites (IRESes), there has been only few studies focusing on the role of cap-independent translation in leukemia and lymphomas. Expression of IRES trans-acting factors can also be altered, and interestingly, BCL-ABL1 fusion protein expressed from "Philadelphia" chromosome, found in some types of leukemia, regulates several of them. A mechanism involving c-Myc IRES and cap-independent translation and leading to resistance to chemotherapy in multiple myeloma emphasize the contribution of cap-independent translation in blood cancers and the need for more work to be done to clarify the roles of known IRESes in pathology and response to chemotherapeutics

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents

Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis.

The RNA exosome is essential for 3? processing of functional RNA species and degradation of aberrant RNAs in eukaryotic cells. Recent reports have defined the substrates of the exosome catalytic domains and solved the multimeric structure of the exosome complex. However, regulation of exosome activity remains poorly characterized, especially in response to physiological stress. Following the observation that cooling of mammalian cells results in a reduction in 40S:60S ribosomal subunit ratio, we uncover regulation of the nuclear exosome as a result of reduced temperature. Using human cells and an in vivo model system allowing whole-body cooling, we observe reduced EXOSC10 (hRrp6, Pm/Scl-100) expression in the cold. In parallel, both models of cooling increase global SUMOylation, leading to the identification of specific conjugation of SUMO1 to EXOSC10, a process that is increased by cooling. Furthermore, we define the major SUMOylation sites in EXOSC10 by mutagenesis and show that overexpression of SUMO1 alone is sufficient to suppress EXOSC10 abundance. Reducing EXOSC10 expression by RNAi in human cells correlates with the 3? preribosomal RNA processing defects seen in the cold as well as reducing the 40S:60S ratio, a previously uncharacterized consequence of EXOSC10 suppression. Together, this work illustrates that EXOSC10 can be modified by SUMOylation and identifies a physiological stress where this regulation is prevalent both in vitro and in vivo

RTN3 Is a Novel Cold-Induced Protein and Mediates Neuroprotective Effects of RBM3.

Cooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling

An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5′UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions

Etude du contrôle de la traduction des isoformes du VEFG-A

Le VEGF A est un facteur angiogénique majeur intervenant dans l'angiogenèse physiologique et pathologique. Son expression est régulée à toutes les étapes de l'expression génique.. Le pré-ARNm est soumis à un épissage alternatif qui permet la synthèse de trois isoformes majeures de 121, 165 et 189 acides aminés. L'ARNm du VEGF A possède deux sites d'entrée interne des ribosomes (IRES) et deux codons alternatifs d'initiation de la traduction. L'initiation de la traduction au codon AUG, sous le contrôle de l'IRES A, entraîne la synthèse de VEGF A sécrété. Le codon CUG, sous le contrôle de l'IRES B, est responsable de la synthèse du L-VEGF. Cette isoforme est maturée par clivage pour générer une molécule de VEGF A sécrétée et un fragment amino-terminal intracellulaire (le N-VEGF). Nous avons voulu identifier par quel mécanisme la séquence exonique du VEGF, issue de l'épissage alternatif, contrôle l'initiation de la traduction. Nous avons mis en évidence une séquence en amont de l'AUG impliquée dans cette régulation. Cette séquence contenant une uORF, nous avons examiné l'effet de sa traduction sur l'expression du VEGF et le mécanisme de traduction de cette uORF. Nos résultats montrent que cette uORF est fonctionnelle et qu'elle est impliquée dans la régulation de l'expression des isoformes du VEGF. Cette uORF est traduite par un mécanisme indépendant de la coiffe et inhibe la synthèse de l'isoforme 121 dans des conditions environnementales non-hypoxiques. L'ensemble de cette étude permet de mieux comprendre les mécanismes de synthèse des différentes isoformes du VEGF dans des processus pathologiques.VEGF A is a major angiogenic factor, which is implicated in both physiologic and pathologic angiogenesis. Regulation of VEGF A expression occurs at multiple levels of gene expression. Alternative splicing of the pre-mRNA gives rise to three major isoforms of 121, 165 and 189 amino acids. The VEGF mRNA contains two internal ribosome entry sites (IRES) and two alternative initiation codons. The IRES A controls the initiation of the translation at the AUG codon, and the upstream IRES B directs the alternative initiation at the in frame CUG codon, generated a high molecular weight VEGF isoform (L-VEGF). The L-VEGF cleavage generates secreted VEGF and an intracellular fragment, the N-VEGF. We first demonstrated that VEGF IRESs are activated in vivo under environmental stress conditions such as hypoxia, in a mice model of hindlimb ischemia. Morever, we try to identify how the exon content of the VEGF mRNA, determined through alternative splicing, controls the alternative translation initiation. We have determined that this regulation seems occurs in a sequence upstream from the AUG. Since this region contains an upstream open reading frame (uORF), we have examined the involvement of its translation. Our results suggest that this uORF is functional and is involved in alternative translation initiation of the VEGF A isoforms. This uORF can be translated by a cap independent mechanism and leads to the inhibition of VEGF 121 isoform in non-hypoxic environmental condition. These data highlight new regulatory element and provide a framework for understanding the regulation mechanism of VEGF isoforms implicated in pathological condition