Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

Maroto, Federico García; Sierra, J M

doi:10.1128/mcb.9.5.2181

Cited by 33 publications

(47 citation statements)

References 47 publications

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“…We cloned sickle, hid and grim 5 0 UTRs into the monocistronic vector containing Firefly luciferase as a reporter (FLuc; Figure 2a) and assayed their translation competence in a cell-free Drosophila-embryo translation system. 16,17 As expected, the uncapped FLuc control mRNA was not translated efficiently in the extracts, whereas the uncapped reporters containing the 5 0 UTR of hid, grim and sickle conferred translation to the reporter at levels equivalent to the m 7 GpppG-capped FLuc control vector (cap-FLuc; Figure 2b). The same was observed for the cap-independent rpr and hsp70 mRNAs, which were used as controls in this experiment.…”

Section: Resultsmentioning

confidence: 95%

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Different modes of translation for hid, grim and sickle mRNAs in Drosophila

et al. 2006

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Protein synthesis is inhibited during apoptosis. However, the translation of many mRNAs still proceeds driven by internal ribosome entry sites (IRESs). Here we show that the 5 0 UTR of hid and grim mRNAs promote translation of uncapped-mRNA reporters in cell-free embryonic extracts and that hid and grim mRNA 5 0 UTRs drive IRES-mediated translation. The translation of capped-reporters proceeds in the presence of cap competitor and in extracts where cap-dependent translation is impaired. We show that the endogenous hid and grim mRNAs are present in polysomes of heat-shocked embryos, indicating that cap recognition is not required for translation. In contrast, sickle mRNA is translated in a cap-dependent manner in all these assays. Our results show that IRES-dependent initiation may play a role in the translation of Drosophila proapoptotic genes and suggest a variety of regulatory pathways.

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Section: Resultsmentioning

confidence: 95%

“…Translation extracts were prepared from 0 to 12 h-old Drosophila embryos as described 16,17 for the indicated times at 251C. Translation extracts from heat-shocked embryos were prepared from pools of 0-12 h-old embryos that have been treated for 45 min at 371C and processed without further recovery.…”

Section: Methodsmentioning

confidence: 99%

Different modes of translation for hid, grim and sickle mRNAs in Drosophila

et al. 2006

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“…This is true for Drosophila embryos [ 161 and wheat germ (Ravel, J.M., personal communication) systems. We found that eIF-2 -PL has no effect on translation of endogenous mRNAs of Drosophila embryo lysates (table 2) when this system is active in initiation because it is strongly inhibited by cap analogues [13] or pactamycin (table 2). Moreover, the translation of globin mRNA in a wheat germ system was also unaffected by the addition of concentrations of eIF-2 -PL that are active in reticulocyte lysates (table 2).…”

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confidence: 94%

“…The preparation of the cell-free extract from mouse liver has been described [12]. Drosophila embryo lysates [13] were the gift of F. Maroto of this laboratory.…”

Section: Preparationsmentioning

confidence: 99%

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Translational inhibition by eIF‐2‐phospholipid complex in mammalian cell‐free systems

Peláez

Haro

1989

FEBS Letters

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The polypeptide chain initiation factor 2 (eIF‐2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin‐supplemented reticulocyte lysates [De Haro et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6711–6715]. This binding is independent of calcium ions and seems to be specific for phosphatidylinositol or phosphatidylserine; phosphatidic and arachidonic acids are inactive. Like α‐subunit‐phosphorylated eIF‐2, eIF‐2·PL traps GEF in a non‐dissociable eIF‐2·PL·GEF complex whereby GEF is no longer able to recycle. Initiation is inhibited when no free GEF is available. Translational inhibition by eIF‐2·PL is rescued by equimolar amounts of eIF‐2·GEF. On the basis of this stoichiometry, we have estimated that reticulocyte lysates contain about 60 pmol of GEF/ml (60 nM) eIF‐2·PL also inhibits translation in cell‐free mouse liver extracts and this inhibition is prevented by reticulocyte eIF‐2·GEF suggesting that GEF also functions in liver. However, the eIF‐2·PL complex does not affect translation in such non‐mammalian eukaryotic systems as wheat germ and Drosophila embryos.

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Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Ferkovich

Dillard

1990

Arch Insect Biochem Physiol

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Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.

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Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

Cited by 33 publications

References 47 publications

Different modes of translation for hid, grim and sickle mRNAs in Drosophila

Different modes of translation for hid, grim and sickle mRNAs in Drosophila

Translational inhibition by eIF‐2‐phospholipid complex in mammalian cell‐free systems

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

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