Studying Translational Control in Drosophila Cell-Free Systems

Centre de Regulació Genómica (CRG-UPF), Barcelona, Spain.
Methods in Enzymology (Impact Factor: 2.09). 02/2007; 429:23-33. DOI: 10.1016/S0076-6879(07)29002-0
Source: PubMed


Classically, Drosophila cell-free translation systems have been used to study the response of the translational machinery to heat shock treatment. We and others have developed optimized Drosophila embryo and ovary extracts, and their use has expanded to the study of a variety of translational control events. These extracts recapitulate many of the aspects of mRNA translation observed in vivo and retain critical regulatory features of several translational control processes. Indeed, their use is rapidly improving our knowledge of molecular mechanisms of translational control. In this chapter we provide general guidelines and detailed protocols to obtain and use translation extracts derived from Drosophila embryos and ovaries.

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    • "Reaper IRES (168 nucleotides) was inserted into the 5 0 UTR of luciferase mRNA. In vitro translation assays were conducted with Drosophila embryo extract, as described previously (Gebauer and Hentze, 2007). Coelentarazine (3 mM final concentration) was added to monitor the time course of luciferase synthesis using a 96-well plate reader (Genios, Tecan). "
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    ABSTRACT: Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. Here, we show that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosome⋅FMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome.
    Molecular cell 04/2014; 54(3). DOI:10.1016/j.molcel.2014.03.023 · 14.02 Impact Factor
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    • "In vitro systems have been instrumental in deciphering mechanisms of translational control (Gebauer and Hentze 2007; Mathews et al. 2007 and references therein; Wu et al. 2007). To analyze PUF-mediated repression in S. cerevisiae, we developed an assay in which recombinant, canonical PUF proteins repressed translation in a yeast cell extract. "
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    ABSTRACT: PUF (Pumilio and FBF) proteins provide a paradigm for mRNA regulatory proteins. They interact with specific sequences in the 3' untranslated regions (UTRs) of target mRNAs and cause changes in RNA stability or translational activity. Here we describe an in vitro translation assay that reconstitutes the translational repression activity of canonical PUF proteins. In this system, recombinant PUF proteins were added to yeast cell lysates to repress reporter mRNAs bearing the 3'UTRs of specific target mRNAs. PUF proteins from Saccharomyces cerevisiae and Caenorhabditis elegans were active in the assay and were specific by multiple criteria. Puf5p, a yeast PUF protein, repressed translation of four target RNAs. Repression mediated by the HO 3'UTR was particularly efficient, due to a specific sequence in that 3'UTR. The sequence lies downstream from the PUF binding site and does not affect PUF protein binding. PUF-mediated repression was sensitive to the distance between the ORF and the regulatory elements in the 3'UTR: excessive distance decreased repression activity. Our data demonstrate that PUF proteins function in vitro across species, that different mRNA targets are regulated differentially, and that specific ancillary sequences distinguish one yeast mRNA target from another. We suggest a model in which PUF proteins can control translation termination or elongation.
    RNA 06/2010; 16(6):1217-25. DOI:10.1261/rna.2070110 · 4.94 Impact Factor
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    ABSTRACT: This paper describes an entrainment model for cohesive sediments that is based on a power-law expression for the excess shear stress and a total entrainment coefficient. Total entrainment includes terms for consolidation. bioturbation, and all other processes (base entrainment coefficient). The model is used to predict entrainment rates for cohesive sediments from Lake Erie, the Tamar Estuary, Long Island Sound, and the Fox River, Wisconsin. The base entrainment coefficient. which is estimated using samples with the least post-depositional modification, is unique for each sediment suite because it includes environmentally sensitive processes like mineralogy, salinity. organic carbon content. etc. Base on available entrainment measurements, expressions are presented for the consolidation and bioturbation coefficients. The model is evaluated with entrainment data for identical sediment that has been either consolidated or bioturbated and the comparison is encouraging. A comparison of predicted and measured entrainment rates for undisturbed sediment is less favorable because of its unknown post-depositional history.
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