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.

Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

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.

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.

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.

In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis: (lens/regeneration/transdifferentiation).

After lentectomy of larval Xenopus laevis, the outer cornea undergoes tissue transformation resulting in formation of a new lens. This lens regeneration is triggered and sustained by neural retina. In the present study, lens-forming transformation of the outer cornea was completed in vitro when the outer cornea was cultured within the lentectomized eye-cup. Well-differentiated lens fiber cells, which showed positive immunofluorescence for total crystallins, were also formed when the outer cornea was cultivated with the retina. No lens tissue was formed when the cornea was cultured alone. Lens-forming transformation, originating from the cornea three and five days after lentectomy, completely regressed when the tissue was isolated in vitro. Fom the present and previous findings, we concluded that, the interaction of corneal cells with the retina plays a decisive role in lens regeneration in situ.