Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase

The SUC2 gene of yeast (Saccharomyces) encodes two forms of invertase: a secreted, glycosylated form, the synthesis of which is regulated by glucose repression, and an intracellular, nonglycosylated enzyme that is produced constitutively. The SUC2 gene has been cloned and shown to encode two RNAs (1.8 and 1.9 kb) that differ at their 5′ ends. The stable level of the larger RNA is regulated by glucose; the level of the smaller RNA is not. A correspondence between the presence of the 1.9 kb RNA and the secreted invertase, and between the 1.8 kb RNA and the intracellular invertase, was observed in glucose-repressed and -derepressed wild-type cells. In addition, cells carrying a mutation at the SNF1 locus fail to derepress synthesis of the secreted invertase and also fail to produce stable 1.9 kb RNA during growth in low glucose. Glucose regulation of invertase synthesis thus is exerted, at least in part, at the RNA level. A naturally silent allele (suc2°) of the SUC2 locus that does not direct the synthesis of active invertase was found to produce both the 1.8 and 1.9 kb RNAs under normal regulation by glucose. A model is proposed to account for the synthesis and regulation of the two forms of invertase: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequence, resulting in synthesis of the intracellular enzyme.     

Polymorphisms in the 3′ UTR in the neurocalcin δ gene affect mRNA stability, and confer susceptibility to diabetic nephropathy

Using a large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese type 2 diabetic patients, we have identified a gene encoding neurocalcin δ (NCALD) as a candidate for a susceptibility gene to diabetic nephropathy; the landmark SNP was found in the 3′ UTR of NCALD (rs1131863: exon 4 +1340 A vs. G, P = 0.00004, odds ratio = 1.59, 95% CI 1.27–1.98). We also discovered two other SNPs in exon 4 of this gene (+999 T/A, +1307 A/G) that showed absolute linkage disequilibrium to the landmark SNP. Subsequent in vitro functional analysis revealed that synthetic mRNA corresponding to the disease susceptible haplotype (exon 4 +1340 G, +1307 G, +999 A) was degraded faster than mRNA corresponding to the major haplotype (exon 4 +1340 A, +1307 A, +999 T), and allelic mRNA expression of the disease susceptibility allele was significantly lower than that of the major allele in normal kidney tissues. In an experiment using a short interfering RNA targeting NCALD, we found that silencing of the NCALD led to a considerable enhancement of cell migration, accompanied by a significant reduction in E-cadherin expression, and by an elevation of α smooth muscle actin expression in cultured renal proximal tubular epithelial cells. We also identified the association of the landmark SNP with the progression of diabetic nephropathy in a 8-year prospective study (A vs. G, P = 0.03, odds ratio = 1.91, 95% CI 1.07–3.42). These results suggest that the NCALD gene is a likely candidate for conferring susceptibility to diabetic nephropathy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.     

Serial production of nucleoside-5′-triphosphates and uracil from 3′-mononucleotides by intact yeast cells

Summary Nucleoside-5-triphosphates such as 5-ATP and 5-GTP can be produced efficiently and continuously from 3-mononucleotides such as 3-AMP and 3-GMP by a series of processes consisting of two reaction phases using dried cells of Candida sp. N-25-2 (a hydrocarbon assimilating yeast). Moreover, incidentally to the 5-triphosphates, free uracil is yielded almost stoichiometrically from 3-CMP and 3-UMP which, as is well known, are main concomitant products depolymerized from RNA. Uracil is then also available for many usage.     

Tombusvirus recruitment of host translational machinery via the 3′ UTR

RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5′-cap structure and a 3′-poly(A) tail, and instead rely on a 3′-cap-independent translational enhancer (3′CITE) located in their 3′ untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 3′CITE in tombusviruses—also present in aureusviruses and carmoviruses—using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 5′ UTR of the viral genome. The specificity of 3′CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 5′ UTR relies on complementary RNA–RNA base-pairing. We show for the first time that this tripartite 5′ UTR/3′CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 5′ end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 3′CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 3′CITE function. Moreover, the virus–host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 3′CITE activity.     

Improved efficiency and stability of secnidazole – An ideal delivery system

Secnidazole (α,2-Dimethyl-5-nitro-1H-imidazole-1-ethanol) is a highly effective drug against a variety of G⁺/G⁻ bacteria but with significant side effects because it is being used in very high concentration. In this study, gold nanoparticles (GNP_S) were selected as a vehicle to deliver secnidazole drug at the specific site with more accuracy which made the drug highly effective at substantially low concentrations. The as-synthesized GNPs were capped with Human Serum Albumin (HSA) and subsequently bioconjugated with secnidazole because HSA provides the stability and improves the solubility of the bioconjugated drug, secnidazole. The quantification of covalently bioconjugated secnidazole with HSA encapsulated on enzymatically synthesized GNPs was done with RP-HPLC having SPD-20 A UV/VIS detector by using the C-18 column. The bioconjugation of GNPs with secnidazole was confirmed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The bioconjugated GNPs were characterized by UV–VIS spectroscopy, TEM, Scanning Electron Microscopy (SEM) and DLS. Zeta potential confirmed the stability and uniform distribution of particles in the emulsion of GNPs. The separation of bioconjugated GNPs, unused GNPs and unused drug was done by gel filtration chromatography. The minimal inhibitory concentration of secnidazole-conjugated gold nanoparticles (Au-HSA-Snd) against Klebsiella pneumonia (NCIM No. 2957) and Bacillus cereus (NCIM No. 2156) got improved by 12.2 times and 14.11 times, respectively, in comparison to pure secnidazole. Precisely, the MIC of Au-HSA-Snd against K. pneumonia (NCIM No. 2957) and B. cereus (NCIM No. 2156) were found to be 0.35 and 0.43 μg/ml, respectively whereas MIC of the pure secnidazole drug against the same bacteria were found to be 4.3 and 6.07 μg/ml, respectively.     

Identification of proteins specifically interacting with YB-1 mRNA 3′ UTR and the effect of hnRNP Q on YB-1 mRNA translation

In this study, proteins specifically interacting with the 3′ untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3′ UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator — YB-1 itself.     

Synthesis and characterization of small interfering RNAs with haloalkyl groups at their 3′-dangling ends

With the aim to create a small interfering RNA (siRNA) with enhanced activity and resistance to nuclease degradation, we synthesized and evaluated the properties of the following siRNAs containing haloalkyl β-d-ribofuranosides at their 3′-dangling ends: 2,2,2-trifluoroethyl β-d-ribofuranoside, 2,2,2-trichloroethyl β-d-ribofuranoside and 2,2,2-tribromoethyl β-d-ribofuranoside. The gene silencing activities of the modified siRNAs were investigated through a dual luciferase reporter assay using HeLa cells. The highest silencing activity was observed for the trichloroethyl analog modified siRNA, which was closely followed by the trifluoroethyl and tribromoethyl analogs. The modified siRNAs were found to show increased binding affinity towards the Piwi-Argonaute-Zwille (PAZ) domain protein based on computational analysis and an experimental study. Furthermore, the RNAs modified with the analogs at their 3′-ends exhibited improved resistance to hydrolysis by a 3′-exonuclease.     

Synthesis of 3′-Fluoro-3′-Deoxyribonucleosides; Anti-HIV-1 and Cytostatic Properties

Abstract

It has generally proven difficult to synthesize ribonucleosides with sugar modifications at the 3′ position. We now present a practical route for the synthesis of ribonucleosides with a 3′ fluorine substituent. Nucleosides with the xylo configuration were prepared by sugar-base condensation. Tritylation of the unprotected nucleosides gave a mixture of 2′,5′ and 3′,5′ bistritylated nucleosides which were difficult to characterize. Therefore the necessary precursors were synthesized in a step-wise fashion, starting with selective deprotection of the 2′-acyl group, followed by tritylation. This gave the 2′,5′-tritylated xylonucleosides in good yield. Reaction with diethylaminosulfur trifluoride and deprotection with 80 % acetic acid provided the 3′-fluoro-3′-deoxyribonucleosides 1, 2 and 4. The cytidine derivative was synthesized from 1 by reaction with trifluoromethanesulfonic anhydride followed by ammonia. Treatment of 4 with adenosine deaminase yielded 5.