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.

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.

The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

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.

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.

Leptin is an adipocyte-derived hormone which acts as a major regulator for food intake and energy homeostasis. Previous studies have focused on the association between polymorphisms in the coding region of the leptin (LEP) pig gene and economically important traits. The present work aimed to study the leptin gene at the promoter region to search for polymorphisms that could lead to different gene expression regulation. In the 1213 bp 5’ gene flanking region, we identified 17 new polymorphisms in two pig breeds characterized by different fat content, namely Casertana and Large White. In order to investigate whether a polymorphism affects the transcriptional activity of the leptin promoter, we evaluated in vitro reporter gene activity driven by two constructs harboring different naturally occurring haplotypes, and we found a two-fold difference in transcription efficiency (P<0.04). These findings may serve as a basis for future studies even in different breeds and might provide molecular data that can be used in association with phenotypic data and finally in the selection scheme.

Telomeres are specialized nucleoprotein structures that have two important functions: (i) protection of the chromosomal ends from deleterious events such as chromosome fusion and degradation; (ii) counteraction of the “end replication problem” by allowing telomerase-dependent or, more rarely, telomerase-independent telomere elongation. The DNA sequences underlying these activities are short simple tandem repeats, which in vertebrate consist of a variable number of TTAGGG. Telomeres dysfunction may be caused either by the absence of telomerase activity or by mutations in telomeric proteins involved in telomere length and structure regulation. Additionally, increasing experimental evidence suggests that telomeres take part in the complex network regulating cell proliferation. Accordingly, telomeres are involved in biological process such as aging and tumor progression. In this study we determined the telomere length in two bovine Italian cattle breeds, Chianina and Maremmana, which are characterized by high longevity and range breeding. In order to account for possible variation among different tissues, we have determined telomere length in different organs such as spleen, lung and liver. Overall, the median telomere length was significant lower in Chianina (11 ± 0.69 kb) than in Maremmana (12.05 ± 1.57 kb). Moreover, telomere length variation among individuals was very low in Chianina but rather high in Maremmana. These data suggest that telomere length is influenced by the breeds. This hypothesis is confirmed by the different history of these Italian breeds. Indeed, Chianina has a long history and its size was maintained by the Breeders Association without necessity to crossbreed with other breeds, whereas the population of Maremmana underwent a dramatic shrinkage in the recent past. Therefore, breeders have crossed Maremmana with other breeds, like Charolais, and have relaxed the rules for the inclusion in the herd book.

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.

Production traits variability among and within breeds, differences among developmental stages or the response to different environments are in part due to genetic factors that affect gene expression. Within the context of an Italian FIRB project, whose objective is to identify genes and molecular mechanisms affecting meat quality and production traits in pig, we studied the promoter regions of candidate genes selected on the basis of their physiological role in animal tissue development or composition. Genomic DNA was isolated from liver or muscle tissue of individuals belonging to Large White and Casertana breed. PCR primers were designed to amplify 5’ upstream region of SCD (Stearoyl-CoA Desaturase), LDLR (Low Density Lipoprotein Receptor), LEP (Leptin), MSTN (Myostatin), ACTA1 (Alpha-actin) and HFABP (Heart Fatty Acid Binding Protein) genes using sequences available at NCBI. A total of 19 single nucleotide polymorphisms (SNPs) not previously described were characterised. Some haplotypes, harbouring SNPs located within, or closely flanking potential cis-acting elements, were used to carry out an in vitro analysis of the efficiency of promoter natural variants. Up to 1200 bases upstream the translation start codon were cloned in pGL3 basic vector in phase with the downstream luciferase reporter gene. The different luminescence intensities showed by constructs harbouring allelic variants suggest transcriptional efficiency influenced by polymorphism. The knowledge of a correlation between a different transcriptional activity and a particular haplotype could be a useful tool to identify variation at genes controlling important traits in pig.

In the present study, the differentiative capacities of Xenopus laevis lens epithelium were tested by isolating it at different stages of development and implanting it autoplastically and homoplastically into the enucleated orbit. The autonomous differentiative capacity of this tissue was also tested in explants cultured in vitro in Leibovitz L 15 with fetal bovine serum added, or serum-free. The results obtained from in vivo experiments showed that whatever were the developmental stages, the eye cup was not necessary for differentiation of lens epithelium into lens fiber cells; this suggested that this tissue has an intrinsic capacity to differentiate into lens. Furthermore, lens epithelium showed an autonomous lens differentiation capacity also when isolated and cultured in vitro, with or without addition of fetal bovine serum.

We investigated the expression of the substance P (SP) receptor (the neurokinin 1 receptor, NK1 receptor) and SP functional effects in developing rabbit retinas. NK1 receptors in adult retinas were in a population of cone bipolar cells and in dopaminergic amacrine cells, as previously described. In contrast, at birth and at postnatal day (PND) 6, NK1 receptors were exclusively expressed by cholinergic amacrine and displaced amacrine cells. NK1 receptor expression in cholinergic cells was still observed at PND10 (eye opening), while at PND21 it was confined to cholinergic cells of the inner nuclear layer. Starting at PND10, NK1 receptors were also in bipolar cells and in dopaminergic amacrine cells. A fully mature NK1 receptor expression pattern was observed at PND35. Dopamine release was assessed in isolated retinas in the presence of SP, the NK1 receptor agonist GR73632 or the NK1 receptor antagonist GR82334. At PND35, extracellular dopamine was significantly increased by 10 microM SP or 0.01-100 microM GR73632, and it was decreased by 0.01-10 microM GR82334. No effects were detected in developing retinas up to PND21. Ca2+ imaging experiments were performed in single cholinergic cells identified by their "starburst" morphology in perinatal retinas. Intracellular Ca2+ levels were significantly increased by 1 microM SP or GR73632. This effect was reversibly inhibited by 1 microM GR82334. These data demonstrate that both NK1 receptor expression and SP physiological actions are developmentally regulated in the retina. SP neurotransmission in the immature retina may subserve developmental events, and SP is likely to represent an important developmental factor for the maturation of retinal neurons and circuitries.

Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.

The outer cornea of larval Xenopus laevis can reprogram cell differentiation when cultured in medium conditioned by X. laevis neural retina (XRCM) or by Rana esculenta neural retina (RRCM). Under these experimental conditions corneal cells showed the same series of cytological changes of fibre cell differentiation observed during ontogenesis and in vivo lens regeneration: enlargement of nuclei and nucleoli, increase of ribosomal population (cytoplasm-basophilia), cell elongation gradual loss of basophilic properties and acquisition of acidophilic properties for crystallin synthesis and accumulation. These events were completely dependent on XRCM or RRCM, suggesting that the neural retina secretes a factor(s) which initiates and sustains lens fibre transdifferentiation of the corneal epithelial cells. This culture system appears to be a suitable one for investigating the control of lens fibre transdifferentiation in vitro.

It has been shown that lens regeneration from outer cornea of larval Xenopus laevis is dependent on neural retina both in vivo and in tissue culture. The isolated outer cornea cultured in the presence of bovine brain-derived acidic Fibroblast Growth Factor (aFGF) is able to reprogram the differentiation into lens fibers, although this transdifferentiative process is not coupled with the formation of a normally organized lens. The capacity of aFGF to promote lens differentiation from cornea is not linked to its mitogenic activity. The cultured corneal cells can transdifferentiate into lens fibers in the presence of aFGF when DNA replication and cell proliferation are prevented by addition of aphidicolin, a specific inhibitor of DNA polymerase in eukaryotes, to the culture medium.

Precedenti ricerche hanno dimostrato che la rigenerazione délia lente dalla cornea esterna di larve di Xenopuslaevis dipende dall’influenza esercitata dalla retina neurale siain situ sia in coltura di tessuto. Nella présente ricerca la cornea esterna di larve diXenopus laevis è stata coltivatain vitro in Leibovitz L 15 diluito, in presenza di siero fetale di vitello e di 500 ng/ml di FGF acido. I risultati ottenuti evidenzian-do che il transdifferenziamento lentogeno della cornea può essere stimolato da aFGF e suggeriscono che tale fattore di crescita potrebbe essere prodotto dalla retina neurale durante la rigenerazione della lente.

Nella presente ricerca l’epitelio della lente di larve diXenopus laevis è stato isolato ed impiantato autoplasticamente al fine di accertare la possibile capacità differenziativa autonoma di questo tessuto. I risultati ottenuti mostrano che il differenziamento deü’epitelio della lente è indipendente dalla presenza del’occhio e suggeriscono quindi 1’esistenza di capacità differenziative autonome di questo tessuto oculare.