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.

Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.

During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRES(CP)). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRES(CP) requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRES(CP) and the 3'-poly(A).

A point mutation in translation initiation factor 2B leads to a continuous hyper stress state in oligodendroglial-derived cells.

BACKGROUND: Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM). The disease is manifested by loss of brain myelin upon physiological stress. In a previous study, we showed that fibroblasts isolated from CACH/VWM patients are hypersensitive to pharmacologically-induced endoplasmic reticulum (ER) stress. Since brain cells from affected individuals are not available for research, we wished to assess the effect of eIF2B mutation on oligodendroglial-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: A rat oligodendroglial-derived cell line was used for a stable knock-down of eIF2B5 followed by stable expression of mutated eIF2B5(R195H) cDNA. In response to a pharmacological ER-stress agent, eIF2B5(R195H) expressing cells exhibited heightened ER-stress response demonstrated by hyper induction of ATF4, GADD34, Bip, PDIA1, PDIA3, PDIA4 and PDIA6 proteins. Moreover, even in the absence of a pharmacological stress agent, eIF2B5(R195H)-expressing cells exhibited high basal levels of ATF4, GADD34 and ER-associated Bip, PDIA1 and PDIA3. SIGNIFICANCE: The data provide evidence that oligodendroglial-derived cells expressing a mutated eIF2B constantly use their stress response mechanism as an adaptation mean in order to survive. The current study is the first to demonstrate the effects of eIF2B5 mutation on ER homeostasis in oligodendroglial-derived cells.

The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation.

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1). We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex.