|
Subcellular Localization and Regulated Translation of mRNA in Drosophila Development
Summary: Robin Wharton is interested in understanding the molecular mechanisms that underlie pattern formation in Drosophila.
In many organisms, the subcellular localization of proteins and mRNAs underlies two superficially different biological processes—the maintenance of stem cell identity and the establishment of embryonic polarity. We have been studying the localization of proteins and mRNAs in two highly polarized cell types during Drosophila development.
Asymmetric Inheritance of a Translational Repressor During Neural Stem Cell Division
Specification of abdominal segments in the early embryo is governed by a repressor complex assembled on the 3'-untranslated region (3'-UTR) of hunchback (hb) mRNA. One component of this complex, Brain Tumor (BRAT), was also known to be involved in neural development; as its name implies, brat– larval brains are grossly enlarged and capable of metastasizing upon transplantation into a wild-type host. To understand better the cellular basis of this poorly restrained growth, we searched for candidate partners of the conserved NHL domain of BRAT that is critical for its function, both as a translational repressor in the early embryo and a growth suppressor in the larval brain.
One intriguing BRAT partner to emerge from these studies is Miranda (MIRA), previously known to play a key role in the asymmetric partitioning of proteins during the division of neural stem cells (neuroblasts). At mitosis, MIRA accumulates in a basal cortical crescent of the neuroblast. Orientation of the spindle ensures that MIRA and MIRA-associated cargo are (transiently) cleared from the self-renewing neuroblast and inherited by the small basal daughter cell that ultimately gives rise to neurons.
We have shown that BRAT is a new MIRA cargo, concentrated in a basal crescent of embryonic and larval neuroblasts at mitosis and subsequently inherited by the basal daughter cell. In collaboration with Chris Doe (HHMI, University of Oregon), we have shown that a key role of BRAT is to ensure that the Prospero transcription factor is loaded onto MIRA and inherited by the basal daughter cell following mitosis.
Germline-Specific Translational Repression of CyclinB mRNA
In addition to its role in regulating abdominal segmentation, Nanos (NOS) is required for many different aspects of primoridal germ cell (PGC) development, where it blocks proliferation, enhances survival, and promotes germline stem cell maintenance.
We have shown that maternal CycB mRNA is directly regulated by binding of Pumilio (PUM) and NOS in concert to an element in its 3'-UTR. To investigate the mechanism by which repression of CycB is limited to the germline, we misexpressed NOS on its own or in the presence of the germline determinant Oskar (OSK); these experiments reveal that, in addition to its other known functions, OSK directs the production or concentration of at least one additional cofactor that cooperates with NOS and PUM to repress CycB mRNA exclusively in the PGCs. A requirement for dual germline-restricted regulators (NOS plus the unidentified cofactor) presumably ensures that CycB is never repressed in the somatic cytoplasm, which we have shown results in mitotic failure and severe developmental defects.
Localization of nos mRNA to the Pole Plasm
A minor fraction of nos mRNA is localized to the pole plasm, where it is the sole source of NOS protein, essential for abdominal segmentation. Despite its importance in both body patterning and germline development, the mechanisms underlying localization of nos mRNA to the posterior of the oocyte are not well defined. Several mRNAs in Drosophila and in other organisms are localized via motor molecules that traverse cytoskeletal elements. In contrast, nos mRNA is trapped in passing by an anchor prelocalized to the posterior cortex as the cytoplasm is churned in a process known as cytoplasmic streaming.
To identify components of the nos mRNA localization machinery, we have performed a genetic screen in a sensitized background in which only barely sufficient NOS is generated to permit abdominal segmentation. One of the genes isolated in this screen encodes the HSP90 (heat-shock protein 90) molecular chaperone, which specializes in stabilizing signaling molecules that fold to a near-native conformation. Compromising HSP90 function specifically affects the localization of nos and pgc mRNAs, which, evidently, comprise a specialized subset of the mRNAs localized late in oogenesis to the posterior pole. Overexpression of the LKB1 kinase largely rescues the localization of pgc mRNA in hsp90 mutant animals, strongly suggesting it is a major HSP90 client for mRNA localization.
Another gene isolated in the screen for localization factors encodes a novel protein with no identifiable motifs. Analysis of genetic mosaics suggests this novel factor may regulate the actin cytoskeleton, to which nos mRNA and the pole plasm ultimately are anchored. Our current studies are focused on understanding where and when this novel gene product acts. We are also studying interacting factors in an effort to understand its biochemical mechanism of action.
Repression of the Unlocalized nos mRNA in the Prospective Somatic Cytoplasm
Repression of the unlocalized nos mRNA found outside the pole plasm during early embryonic development is mediated by binding of Smaug (SMG) to sites in the 3'-UTR. The RNA-binding domain of SMG interacts with the eIF-4E binding protein Cup; this interaction has been proposed to be responsible for at least part of the repressor activity of SMG. The RNA-binding domain also interacts with OSK, suggesting that OSK acts directly to relieve SMG-dependent repression in the pole plasm. We are testing these ideas for the mechanism of SMG action by identifying mutant derivatives that do not interact with Cup or OSK. We are also studying the incorporation of SMG into the CCR4/POP2/NOT deadenylase complex, which is a central player in regulation of mRNA stability in Drosophila and other organisms.
Work on the role of BRAT was supported by a grant from the National Institutes of Health.
|
