Smaug triggers destabilization and localization of specific maternal transcripts through recruitment of the CCR4/POP2/NOT deadenylase. In contrast, Smaug-mediated translational repression is accomplished via an indirect interaction between Smaug and eIF4E, a component of the basic translation machinery. Thus, Smaug is a multifunctional posttranscriptional regulator that employs distinct mechanisms to repress translation and to induce degradation of target transcripts.

SMAUG (SMG) is an RNA-binding protein that functions as a key component of a transcript degradationpathway that eliminates maternal mRNAs in the bulk cytoplasm of activated Drosophila melanogaster eggs. We previously showed that SMG destabilizes maternal Hsp83 mRNA by recruiting the CCR4-NOT deadenylase to trigger decay; however, the cis-acting elements through which this was accomplished were unknown. Here we show that Hsp83 transcript degradation is regulated by a major element, the Hsp83 mRNA instability element (HIE), which maps to a 615-nucleotide region of the open reading frame (ORF). The HIE is sufficient for association of a transgenic mRNA with SMG protein as well as for SMG-dependent destabilization. Although the Hsp83 mRNA is translated in the early embryo, we show that translation of the mRNA is not necessary for destabilization; indeed, the HIE functions even when located in an mRNA's 3 untranslated region. The Hsp83 mRNA contains eight predicted SMG recognition elements (SREs); all map to the ORF, and six reside within the HIE. Mutation of a single amino acid residue that is essential for SMG's interaction with SREs stabilizes endogenous Hsp83 transcripts. Furthermore, simultaneous mutation of all eight predicted SREs also results in transcript stabilization. A plausible model is that the multiple, widely distributed SREs in the ORF enable some SMG molecules to remain bound to the mRNA despite ribosome transit through any individual SRE. Thus, SMG can recruit the CCR4-NOT deadenylase to trigger Hsp83 mRNA degradation despite the fact that it is being translated.

No abstract

Proteolipid protein (PLP or lipophilin) is a highly conserved, strongly hydrophobic, integral membrane protein, and is the major protein component of central nervous system myelin. Although PLP has been implicated in many functions, its in vivo role is still uncertain. Here, we report the investigation of PLP's putative adhesive function using purified PLP and reconstituted phospholipid vesicles made of either 100% phosphatidylcholine (PC), or a mixture of 92% PC and 8% phosphatidylserine (PS), by weight. PLP-induced changes in the phospholipid bilayer surfaces were directly examined by transmission electron microscopy. We found that upon the introduction of PLP, larger lipid vesicles became smaller and unilamellar. At the PLP:lipid molar ratio of 1:20, vesicle membranes rolled onto themselves forming 'croissant'-like structures that subsequently adhered to each other. The phenomena of PLP-induced bilayer rolling and adhesion were dependent on the concentration of PLP and the period of incubation, but were independent of the presence of calcium and types of phospholipids (PC or PC:PS). Furthermore, the presence of PLP in the lipid bilayers prevented the fusion of membranes. These findings show that PLP can induce membrane 'winding' while preventing the fusion of adjacent lipid bilayers. Hence, our data provide direct evidence for PLP's suspected function of membrane adhesion, and also suggest that PLP could potentially play a role in the formation of the myelin sheath.

No abstract

In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation.

Transmission electron microscopy and single particle electron crystallography were employed to reconstruct high-quality projection images of a recombinant, acidic tail deficient form of rat upstream binding factor. The upstream binding factor was found to be dimeric and approximately 10 nm in diameter with a central region of low density. Distinct nodes were observable, of size and spacing consistent with being HMG boxes 3 and 4. The dimerisation domain seemed most probably to be located in the internal region of the structure.

Transmission electron microscopy and single particle electron crystallography were employed to reconstruct high-quality projection images of a recombinant, acidic tail deficient form of rat upstream binding factor. The upstream binding factor was found to be dimeric and approximately 10 nm in diameter with a central region of low density. Distinct nodes were observable, of size and spacing consistent with being HMG boxes 3 and 4. The dimerisation domain seemed most probably to be located in the internal region of the structure.