Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicDA40V protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicDA40V function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser103. Remarkably, amongst the Drosophila serines we found phosphorylated, Ser103 is the only one that is fully conserved in mammalian BicD

Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

Post-transcriptional gene regulation: From genome-wide studies to principles

Post-transcriptional regulation of gene expression plays important roles in diverse cellular processes such as development, metabolism and cancer progression. Whereas many classical studies explored the mechanistics and physiological impact on specific mRNA substrates, the recent development of genome-wide analysis tools enables the study of post-transcriptional gene regulation on a global scale. Importantly, these studies revealed distinct programs of RNA regulation, suggesting a complex and versatile post-transcriptional regulatory network. This network is controlled by specific RNA-binding proteins and/or non-coding RNAs, which bind to specific sequence or structural elements in the RNAs and thereby regulate subsets of mRNAs that partly encode functionally related proteins. It will be a future challenge to link the spectra of targets for RNA-binding proteins to post-transcriptional regulatory programs and to reveal its physiological implications

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Abstract Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction

THEORETICAL STUDY OF THE ELECTRONIC STATES WITH SPIN-ORBIT EFFECTS AND ROVIBRATIONAL CALCULATIONS OF THE MOLECULE LiCs

Author Institution: Faculty of Science, Beirut Arab University, P.O. Box 11-5020 Riad El Solh, Beirut, 1107 2809, Lebanon.Due to the advancement in the ultracold alkali atom trapping developments, which are at the root of photoassociation spectroscopy, the potential energy curves have been calculated for the 82 lowest electronic states of the molecule LiCs including the spin orbit effect within the range 3.0a$_{o}$ to 28.0a$_{o}$ of the internuclear distance R for the symmetries $^{1,3}\Sigma$% , $^{1,3}\Pi$, $^{1,3}\Delta$, and $\Omega$=0$^{-}$, 0$^{+}$, 1, 2, 3 along with the spectroscopic constants for 66 electronic states. To investigate the electronic structure of these electronic states, the atoms Li and Cs have been treated through non-empirical relativistic effective one-electron core potential of the Durand and Barthelat type. Gaussian basis sets have been used in this calculation for the Li and Cs atoms. Core valence effects including core-polarization and core valence correlation are taken into account by using an l-dependent core-polarization potential. In order to reproduce the experimental splitting with a very good agreement for Li and Cs, semi-empirical spin orbit pseudo-potential has been designed for these atoms. The molecular orbitals of the molecule LiCs have been derived from self consistent field (SCF) calculation and full valence configuration interaction (CI) calculations were performed. The core-core interaction calculations are performed as the Hartree-Fock energy of the ion (LiCs)$$. The calculations were performed in a pseudo-potential scheme. To investigate the electronic structure including the spin-orbit effects (SO) we used the package CIPSO which allows a full CI calculation as well as perturbative CI calculations with SO effects. Using the canonical functions approach, the values of the eigenvalue E$_{v}$, the abscissas of the turning points (R$_{min}$, R$_{max}$), the rotational constants B$_{v}$, and the centrifugal distortion constants D$_{v}$ have been calculated up to 76 vibrational levels for 20 states. Through the same approach, the dipole moment functions have been calculated for most of the states in the $\Lambda$-representation along with the vibrational matrix elements for the states (1)$^{1}\Sigma ^{+}$, (1)$^{3}\Sigma ^{+}$, (1)$^{1}{\Pi }$, and (1)^{3}{% \Pi }. The comparison of the present results with those available in the literature shows a very good agreement

Use of XPS to detect variations in dispersion of impregnated and ion-exchanged NiO/SiO/sub 2/ systems

The use of XPS intensity measurements to follow the dispersion of impregnated and ion-exchanged NiO/SiO/sub 2/ samples has been examined. Intensity measurements clearly demonstrate the profound difference in dispersion between the two series. Moreover, as revealed by comparison with independent dispersion measurements, they adequately predict the evolution of the dispersion of NiO as a function of nickel content in the impregnated series.Anglai

The dispersion of nickel oxide supported on boron-modified silicas, as determined by TEM and XPS measurements

The dispersion of NiO supported on boron-modified silicas has been studied by transmission electron microscopy (TEM) and XPS. The TEM experiments showed that all NiO is present in the form of crystallites; values for the dispersion were calculated on the basis of measurements of the size distributions of these crystallites. The XPS measurements were interpreted using a model which assumes large particle sizes and no re-partitioning of the supported species between internal pores and the surface when boron-modification is performed. The variations of dispersion with boron content obtained from this model agree well with the variations measured by TEM.Anglai