A Screen for RNA-Binding Proteins in Yeast Indicates Dual Functions for Many Enzymes

Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation

Yeast Mitochondrial Biogenesis: A Role for the PUF RNA-Binding Protein Puf3p in mRNA Localization

The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis

Characteristics of transposable element exonization within human and mouse

Insertion of transposed elements within mammalian genes is thought to be an important contributor to mammalian evolution and speciation. Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Elucidation of the evolutionary constraints that have shaped fixation of transposed elements within human and mouse protein coding genes and subsequent exonization is important for understanding of how the exonization process has affected transcriptome and proteome complexities. Here we show that exonization of transposed elements is biased towards the beginning of the coding sequence in both human and mouse genes. Analysis of single nucleotide polymorphisms (SNPs) revealed that exonization of transposed elements can be population-specific, implying that exonizations may enhance divergence and lead to speciation. SNP density analysis revealed differences between Alu and other transposed elements. Finally, we identified cases of primate-specific Alu elements that depend on RNA editing for their exonization. These results shed light on TE fixation and the exonization process within human and mouse genes.Comment: 11 pages, 4 figure

Analysis of Microsatellite Variation in Drosophila melanogaster with Population-Scale Genome Sequencing

Genome sequencing technologies promise to revolutionize our understanding of genetics, evolution, and disease by making it feasible to survey a broad spectrum of sequence variation on a population scale. However, this potential can only be realized to the extent that methods for extracting and interpreting distinct forms of variation can be established. The error profiles and read length limitations of early versions of next-generation sequencing technologies rendered them ineffective for some sequence variant types, particularly microsatellites and other tandem repeats, and fostered the general misconception that such variants are inherently inaccessible to these platforms. At the same time, tandem repeats have emerged as important sources of functional variation. Tandem repeats are often located in and around genes, and frequent mutations in their lengths exert quantitative effects on gene function and phenotype, rapidly degrading linkage disequilibrium between markers and traits. Sensitive identification of these variants in large-scale next-gen sequencing efforts will enable more comprehensive association studies capable of revealing previously invisible associations. We present a population-scale analysis of microsatellite repeats using whole-genome data from 158 inbred isolates from the Drosophila Genetics Reference Panel, a collection of over 200 extensively phenotypically characterized isolates from a single natural population, to uncover processes underlying repeat mutation and to enable associations with behavioral, morphological, and life-history traits. Analysis of repeat variation from next-generation sequence data will also enhance studies of genome stability and neurodegenerative diseases

Efficacy of thalidomide in a girl with inflammatory calcinosis, a severe complication of juvenile dermatomyositis

We report a 14-year-old girl with juvenile dermatomyositis (JDM) complicated by severe inflammatory calcinosis successfully treated with thalidomide. She was diagnosed as JDM when she was 4 years old after a few months of increasing lethargy, muscle pain, muscle weakness, and rash. During three months, clinical manifestations and abnormal laboratory findings were effectively treated with oral prednisolone. However, calcinosis was recognized 18 months after disease onset. Generalized calcinosis rapidly progressed with high fever, multiple skin/subcutaneous inflammatory lesions, and increased level of CRP. Fifty mg/day (1.3 mg/kg day) of oral thalidomide was given for the first four weeks, and then the dose was increased to 75 mg/day. Clinical manifestations subsided, and inflammatory markers had clearly improved. Frequent high fever and local severe pain with calcinosis were suppressed. The levels of FDP-E, IgG, and tryglyceride, which were all elevated before the thalidomide treatment, were gradually returned to the normal range. Over the 18 months of observation up to the present, she has had no inflammatory calcinosis, or needed any hospitalization, although established calcium deposits still remain. Her condition became painless, less extensive and less inflammatory with the CRP level below 3.08 mg/dL. Recent examination by whole-body 18F-FDG-PET-CT over the 15 months of thalidomide treatment demonstrated fewer hot spots around the subcutaneous calcified lesions

Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids

Plasma therapy in atypical haemolytic uremic syndrome: lessons from a family with a factor H mutation

Whilst randomised control trials are undoubtedly the best way to demonstrate whether plasma exchange or infusion alone is the best first-line treatment for patients with atypical haemolytic uremic syndrome (aHUS), individual case reports can provide valuable information. To that effect, we have had the unique opportunity to follow over a 10-year period three sisters with aHUS associated with a factor H mutation (CFH). Two of the sisters are monozygotic twins. A similar natural evolution and response to treatment would be expected for the three patients, as they all presented with the same at-risk polymorphisms for CFH and CD46 and no identifiable mutation in either CD46 or CFI. Our report of different modalities of treatment of the initial episode and of three transplantations and relapses in the transplant in two of them, strongly suggest that intensive plasma exchange, both acutely and prophylactically, can maintain the long-term function of both native kidneys and allografts. In our experience, the success of plasma therapy is dependent on the use of plasma exchange as opposed to plasma infusion alone, the prolongation of daily plasma exchange after normalisation of haematological parameters followed by prophylactic plasma exchange, the use of prophylactic plasma exchange prior to transplantation and the use of prophylactic plasma exchange at least once a week posttransplant with immediate intensification of treatment if there are any signs of recurrence

Donation Crowdfunding : Principles and Donor Behaviour

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Reliability, Validity and Psychometric Properties of the Greek Translation of the Center for Epidemiological Studies-Depression (CES-D) Scale

INTRODUCTION: The aim of the current study was to assess the reliability, validity and psychometric properties of the Greek translation of the Center for Epidemiological Studies- Depression Scale (CES-D). METHODS: 40 depressed patients 29.65 ± 9.38 years old, and 120 normal controls 27.23 ± 10.62 years old entered the study. In 20 of them (12 patients and 8 controls) the instrument was re-applied 1-2 days later. Translation and Back Translation was made. Clinical Diagnosis was reached by consensus of two examiners with the use of the SCAN v.2.0 and the IPDE. Statistical Analysis included ANOVA, the Pearson Product Moment Correlation Coefficient, Principal Components Analysis and Discriminant Function Analysis and the calculation of Cronbach's alpha (α) RESULTS: Both Sensitivity and specificity exceed 90.00 at 23/24, Chronbach's alpha for the total scale was equal to 0.95. Factor analysis revealed three factors (positive affect, irritability and interpersonal relationships, depressed affect and somatic complains). The test-retest reliability was satisfactory (Pearson's R between 0.45 and 0.95 for individual items and 0.71 for total score). CONCLUSION: The Greek translation of the CES-D scale is both reliable and valid and is suitable for clinical and research use with satisfactory properties. Its properties are similar to those reported in the international literature. However one should always have in mind the limitations inherent in the use of self-report scales