Localisation of RNA

Asymmetry along the anterior-posterior axis of early Drosophila embryos originates from localized mRNAs at both poles of the freshly laid egg. This polar distribution of mRNAs is translated into opposing protein gradients essential for proper embryonic development.

Localization of bicoid (left) and nanos (right) mRNAs in the early Drosophila embryo

Localization of bicoid RNA

bicoid (bcd) mRNA localization is a complicated multistep process, which depends on the 3’UTR of the mRNA and an intact and polarized microtubule cytoskeleton. bcdbcd mRNA is localized in a ring-like fashion at the anterior cortex of the oocyte, but it is later redistributed into a disc-like pattern. The mRNA remains tightly localized at the anterior cortex until oogenesis is completed and the egg is laid.

Localization of bcd mRNA (red in upper panel) during oogenesis, revealed by in situ hybridyzation

Classical genetics identified three maternal genes (exuperantia, swallow and staufen) that are necessary for bcd mRNA localization at different steps. In exuperantiaexu) mutants, bcd mRNA reaches the oocyte, but is not localized at its anterior pole. The biochemical function of Exu protein is not known but it is thought to be involved in the formation of ribonucleoprotein particles. In swallowswa) mutant oocytes, the early bcd mRNA localization steps are normal, but the transition from a ring into a disc-shaped pattern fails to occur and anterior bcd mRNA localization is subsequently lost. Swa binds to the light chain subunit of the Dynein complex, which might be involved in transporting Swa and bcd mRNA to the anterior pole. Staufen protein is a double-stranded RNA binding protein that is required for anchoring bcd mRNA at the anterior cortex during late stages of oogenesis.

Identification of new components in the bcd RNA localization pathway

Many factors essential for bcd RNA localization might be involved in zygotic development or might lead to an early arrest during development, thereby escaping identification in classical female-sterile screens. To overcome these limitations, we performed a genetic screen using the Flp-FRT system, which allows the identification of lethal mutations, in combination with an in situ hybridization assay. We identified several new mutants with defects in bcd RNA localization that we are currently characterizing.

The role of the γ-tubulin ring complex in bcd RNA localization

In our screen, we were able to genetically identify components of the cytoskeleton as essential factors for bcd RNA localization. Mutations in the γ-tubulin ring complex (γTuRC) components γTub37C, Dgrip75 and Dgrip128 lead to bcd RNA localization defects in mid-oogenesis, which are similar to the defect observed in swa mutants. The γTuRC caps microtubule minus ends and serves as a template for microtubule nucleation. Interestingly, γTub37C, Dgrip75 and Dgrip128 mutants have specific defects in bcd RNA localization whereas other microtubule-dependent processes such as osk RNA localization to the posterior pole and nuclear migration are not disrupted. γTub37C, Dgrip75 and Dgrip128 are enriched at the anterior cortex at mid-oogenesis and might organize a subset of microtubules essential for bcd RNA localization. Analysis of additional processes that depend on Dgrip75 and Dgrip128, such as spermatogenesis or female meiosis, revealed that Dgrip75 and Dgrip128 are not essential for microtubule nucleation but rather that they anchor microtubules at specialized microtubule-organizing centers in the male and female germline

Vps36 and bcd RNA localization

In addition we identified the Drosophila homologues of Vps36 and Vps22 as factors essential for bcd mRNA localization. The function of both is required for the late, Staufen-dependent, localization and/or anchoring of bcdbcd 3’UTR in vitro and that the protein co-localizes with the RNA in vivo. In fact, Vps36 is the first protein identified that binds to bcd mRNA in a sequence-specific manner

mRNA. We could show that Vps36, which together with Vps22 and Vps25 forms the highly conserved ESCRT-II complex, binds directly to the

in vitro binding assay (left) and in vivo localization of Vps36 (right) demonstrate direct binding and co-localization of the protein with bcd mRNA

Given the very high homology between Drosophila Vps36 and the human protein (72% amino acid similarity), it is tempting to speculate that the RNA binding activity might be a conserved function of the whole ESCRT-II complex. Therefore we want to study the Drosophila system in more detail; a better understanding of the mechanism of interaction between the Drosophila Vps36 protein and bcd

mRNA might reveal novel functions of the human complex.

Future plans

In the future we would like to continue using the powerful tools of genetics in Drosophila. The Flp-FRT system in combination with a method to directly visualize RNAs in living oocytes should lead to the identification of further genes whose products are involved in bcd mRNA localization. As a complementary approach we plan to biochemically purify complexes containing bcd mRNA and to identify the proteins in these complexes. Together these approaches will further our understanding of mRNA localization within cells and the establishment of polarity and morphogen gradients in embryos and tissues.

Selected Publications

• Irion, U. and St Johnston, D. (2007): bicoid RNA localization requires specific binding of an endosomal sorting complex. Nature 445: 554-558.
  
• Schnorrer, F., Luschnig, S., Koch, I., and Nüsslein-Volhard, C. (2002). γ-Tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during Drosophila oogenesis. Developmental Cell, 3: 685-696.
• Schnorrer, F., Bohmann, K. and Nüsslein-Volhard, C. (2000). Involvement of the dynein molecular motor in targeting Swallow and bicoid RNA to the anterior pole of the Drosophila oocyte. Nature Cell Biology, 2: 185-190.
• Wilsch-Bräuninger, M., Schwarz, H. and Nüsslein-Volhard, C. (1997). A sponge-like structure involved in the association of maternal products during DrosophilaJ. Cell Biol., 139: 817-29.
• Ferrandon, D., Koch, I., Westhof, E. and Nüsslein-Volhard, C. (1997). RNA-RNA interaction is required for the formation of specific bicoid mRNA 3' UTR-STAUFEN ribonucleoprotein particles. EMBO Journal, 16: 1751-8.
• Ferrandon, D., Elphick, L., Nüsslein-Volhard, C. and St Johnston, D. (1994). Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell, 79: 1221-32.

Scientists involved

• Dr. Uwe Irion                                             Project leader (02/06 - )
  
• Ines Wolff                                                  PhD student (07/06 - )
• Alessandra Romano                              PhD student (09/09 - )
  
• (Dr. Nina Vogt; present address: NYU, New York, USA)
  
• (Dr. Frank Schnorrer; present address: IMP, Vienna, Austria)
• (Dr. Dominique Ferrandon; present address: Institut de Biologie Moleculaire et cellulaire, Strasbourg)
• (Dr. Michaela Wilsch-Bräuninger; present address: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden)

Technicians

• Silke Geiger-Rudolph