Ribosomal RACK1 Regulates the Dendritic Arborization by Repressing FMRP Activity.

FMRP is an RNA-binding protein that represses the translation of specific mRNAs. In neurons, its depletion determines the exaggerated translation of mRNAs leading to dendritic and axonal aberrant development, two peculiar features of Fragile X syndrome patients. However, how FMRP binds to translational machinery to regulate the translation of its mRNA targets is not yet fully understood. Here, we show that FMRP localizes on translational machinery by interacting with the ribosomal binding protein, Receptor for Activated C Kinase 1 (RACK1). The binding of FMRP to RACK1 removes the translational repressive activity of FMRP and promotes the translation of PSD-95 mRNA, one specific target of FMRP. This binding also results in a reduction in the level of FMRP phosphorylation. We also find that the morphological abnormalities induced by Fmr1 siRNA in cortical neurons are rescued by the overexpression of a mutant form of RACK1 that cannot bind ribosomes. Thus, these results provide a new mechanism underlying FMRP activity that contributes to altered development in FXS. Moreover, these data confirm the role of ribosomal RACK1 as a ribosomal scaffold for RNA binding proteins.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

BACKGROUND: Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. RESULTS: We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells. CONCLUSIONS: In vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

eIF3d specialized translation requires a RACK1-driven eIF3d binding to 43S PIC in proliferating SH-SY5Y neuroblastoma cells.

Translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eukaryotic initiation factor 4E (eIF4E). Notably, most mRNAs are still capped when eIF4E is inhibited, suggesting alternative mechanisms likely mediate cap-dependent mRNA translation without functional eIF4F. Here we found that, when eIF4E is inhibited, the ribosomal scaffold RACK1 recruits eIF3d on the 43S pre-initiation complex. Moreover, we found that it is just PKCBII in its active form that promotes the binding of RACK1 to eIF3d. These studies disclose a previously unknown role of ribosomal RACK1 for eIF3d specialized translation.

Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells.

BACKGROUND: Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. METHODS: Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. RESULTS: Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. CONCLUSIONS: We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis.

Scientometric and Methodological Analysis of the Recent Literature on the Health-Related Effects of Tomato and Tomato Products.

The health benefits of tomato, a vegetable consumed daily in human diets, have received great attention in the scientific community, and a great deal of experiments have tested their utility against several diseases. Herein, we present a scientometric analysis of recent works aimed to estimate the biological effects of tomato, focusing on bibliographic metadata, type of testers, target systems, and methods of analysis. A remarkably variable array of strategies was reported, including testers obtained by standard and special tomatoes, and the use of in vitro and in vivo targets, both healthy and diseased. In vitro, 21 normal and 36 cancer human cell lines derived from 13 different organs were used. The highest cytotoxic effects were reported on cancer blood cells. In vivo, more experiments were carried out with murine than with human systems, addressing healthy individuals, as well as stressed and diseased patients. Multivariate analysis showed that publications in journals indexed in the agriculture category were associated with the use of fresh tomatoes; conversely, medicine and pharmacology journals were associated with the use of purified and formulate testers. Studies conducted in the United States of America preferentially adopted in vivo systems and formulates, combined with blood and tissue analysis. Researchers in Italy, China, India, and Great Britain mostly carried out in vitro research using fresh tomatoes. Gene expression and proteomic analyses were associated with China and India. The emerging scenario evidences the somewhat dichotomic approaches of plant geneticists and agronomists and that of cell biologists and medicine researchers. A higher integration between these two scientific communities would be desirable to foster the assessment of the benefits of tomatoes to human health.

Novel phenotypes related to the breeding of purple-fruited tomatoes and effect of peel extracts on human cancer cell proliferation.

The production of anthocyanins in the tomato (Solanum lycopersicum L.) fruit is normally absent or poor, but a number of mutants or introgression lines are known to increase anthocyanin levels in vegetative and reproductive tissues. Through conventional breeding, a genetic combination was obtained with the remarkable phenotype of a deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peel. Such a genotype was named Sun Black (SB) as a consequence of its sensitivity to light induction. When characterized for morpho-agronomic traits, SB plants showed increased fertility. Purple fruits displayed an arrangement of the epicarp cells different from normal tomatoes, a feature that could account for different mechanical properties and shelf-life potential. The SB genotype and, to a lesser extent, its single mutant parents showed the capacity to accumulate anthocyanins in the seedling root when grown under light. This phenotype, which was greatly improved by the addition of sucrose to the germination medium, proved to be useful as selection index and gave new insights for in vitro production of anthocyanin extracts. To assess the nutraceutical potential of purple tomatoes, we tested the activity of SB skin extracts on the proliferation of two human cancer cells lines. Cell proliferation was significantly inhibited by SB extract in a dose-dependent manner. When the bioactivity of SB extracts was compared with that of other anthocyanin-containing fruits or vegetables, a significant "Extract*Line" interaction was evidenced, suggesting a crucial role for the extract composition in terms of anthocyanidins and other eventual cell growth-inhibiting compounds.

Design and validation of siRNAs and shRNAs.

RNAi is a highly conserved intracellular mechanism, whereby dsRNA strands conduct post-transcriptional modulation of gene expression through a degradation or inhibition of the translation of target mRNA. Since its discovery in 1998, RNAi has been identified in many different organisms, including mammals, and this mechanism has provided new approaches for studies in cellular and molecular biology, functional genomics and drug discovery. siRNAs can be predicted by sequence and thermodynamic features, and the wide and proficient application of RNAi relies on the ability to select the most active siRNAs from among numerous predicted molecules. Recently, the first-generation prediction algorithms based on the characteristics of siRNAs, short hairpin (sh)RNAs and micro-(mi)RNAs have been improved by the use of computational models that account for the experimentally determined activities of large numbers of siRNAs/shRNAs and miRNAs. These second-generation algorithms differ from the first-generation algorithms in the computational tools that are used for the prediction of siRNA efficacy; although these new algorithms improve the design of effective siRNAs, they do not eliminate the requirement for an experimental evaluation of the activities of siRNAs. This review reports on the most significant second-generation algorithms of siRNA and shRNA characteristics, as well as on recently designed systems for the experimental evaluation of siRNA activities.