Eukaryotic mRNAs possess a 5-terminal cap structure (cap), m 7 GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.

Prions are transmissible agents caused by the self-propagating conformational change of proteins (32). Prions appear to be amyloid protein aggregates that propagate by capturing soluble proteins and converting them into an infectious aggregated form (33). According to the "protein only" hypothesis (32), the prion protein (PrP) is the sole agent responsible for causing numerous infectious diseases, including scrapie (sheep), bovine spongiform encephalopathy (cow), and chronic wasting disease (deer and elk) as well as kuru and Creutzfeld-Jacob disease (humans Het-s] in Podospora anserina, have also been characterized as non-Mendelian inheritable elements (7,37,43). Molecular and genetic studies of these fungal prions have greatly facilitated the elucidation of the molecular basis for prion conversion and propagation as well as the general criteria for prionogenicity in a protein's primary structure.[PSI ϩ ] is a prion form of Sup35, which is the eRF3 polypeptide release factor that is essential for terminating protein synthesis at stop codons (39, 45; for a review, see reference 17). When Sup35 is in the [PSI ϩ ] state, ribosomes often fail to release polypeptides at stop codons, causing a non-Mendelian trait to appear that is easily detected by nonsense suppression (23,29,30). To uncover host factors responsible for [PSI ϩ ] propagation, we have developed a genome-wide genetic selection method for [PSI ϩ ]-eliminating factors or mutants by use of the chromosomal ura3-197 mutant (21). Based on this selection system, we have selected host factors whose high-level expression on a multicopy plasmid leads to [PSI ϩ ] elimination. One clone yielded Rnq1⌬100, an N-terminal truncation of Rnq1, and is further examined in this study. Although there are some reports that the maintenance or de novo appearance of one prion is affected by several genetic manipulations such as overexpression of its own prion domain (15, 16), heterologous prion variants (5, 35), or nonprion protein mutants (1), the molecular basis of the action of one prion in inhibiting heterologous prions is not known.Rnq1 is a protein of unknown function and is one of several known yeast proteins containing a QN-rich prion domain, where the name derives from "rich in asparagine (N) and glutamine (Q)" (37 . According to the seeding model, a heterologous preexisting protein in the prion conformation is used as a template for the conversion of Sup35 into its prion form, which then proceeds to seed its own rapid and separate aggregation. Importantly, [PIN ϩ ] also facilitates the de novo appearance of the prion [URE3] and promotes polyglutamine (polyQ) aggregation and toxicity in general (5,25,27). Therefore, the seeding model predicts that [PIN ϩ ] aggregates provide a "friendly" nidus on which the first seeds of a heterologous prion or polyQ amyloid can form (11,41). The alternative titration model postulates that preexisting heterologous prions or prion-like aggregates capture and inactivate an inhibitor that prevents conversion of Sup35 into a

NeuroD/BETA2, a transcription factor of the insulin gene, also plays an important role in the development of pancreatic beta-cells. Recently, the NeuroD/BETA2 gene has been mapped to the long arm of human chromosome 2 (2q32) where the IDDM7 gene has previously been mapped, implying its involvement in diabetes. To identify mutations in the NeuroD/BETA2 gene that may predispose patients to develop diabetes, we studied the gene in 50 Japanese subjects with diabetes (4 with type 1 and 46 with type 2) by the polymerase chain reaction (PCR) followed by single-strand conformation polymorphism and sequencing analyses. Further analysis was performed in 392 Japanese subjects (60 with type 1 and 158 with type 2 diabetes and 174 healthy control subjects) by mismatch PCR restriction fragment length polymorphism. We found a DNA polymorphism of the NeuroD/BETA2 gene. A nucleotide G-to-A transition results in the substitution of alanine to threonine at codon 45 (Ala45Thr). The frequencies of heterozygotes for the Ala45Thr variant were 9.8% in the control subjects, 9.5% in the patients with type 2 diabetes, and 25.0% in the patients with type 1 diabetes, a significant difference (P = 0.006). Because the variant of the NeuroD/BETA2 gene (Ala45Thr) is associated with type 1 but not type 2 diabetes, it may be implicated in the loss of pancreatic beta-cells in type 1 diabetes.

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer’s disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.

[PIN(+)] is the prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. The function of Rnq1, however, is little understood. The limited availability of defective rnq1 alleles impedes the study of its structure-function relationship by genetic analysis. In this study, we isolated rnq1 mutants that are defective in the stable maintenance of the [PIN(+)] prion. Since there is no rnq1 phenotype available that is applicable to a direct selection or screening for loss-of-function rnq1 mutants, we took advantage of a prion inhibitory agent, Rnq1Delta100, to develop a color-based genetic screen. Rnq1Delta100 eliminates the [PSI(+)] prion in the [PIN(+)] state but not in the [pin(-)] state. This allows us to find loss-of-[PIN(+)] rnq1 mutants as white [PSI(+)] colonies. Nine rnq1 mutants with single-amino-acid substitutions were defined. These mutations impaired the stable maintenance of [PIN(+)] and, as a consequence, were also partially defective in the de novo induction of [PSI(+)]. Interestingly, eight of the nine alleles were mapped to the N-terminal region of Rnq1, which is known as the non-prion domain preceding the asparagine and glutamine rich prion domain of Rnq1. Notably, overexpression of these rnq1 mutant proteins restored [PIN(+)] prion activity, suggesting that each of the rnq1 mutants was not completely inactive. These findings indicate that the N-terminal non-prion domain of Rnq1 harbors a potent activity to regulate the maintenance of the [PIN(+)] prion.