The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.
- MeSH
- buněčný cyklus genetika MeSH
- Drosophila melanogaster genetika MeSH
- ekdyson genetika MeSH
- kultivované buňky MeSH
- larva genetika MeSH
- mutace genetika MeSH
- prekurzory RNA genetika MeSH
- proteiny Drosophily genetika MeSH
- ribonukleoproteiny malé jaderné genetika MeSH
- sestřih RNA genetika MeSH
- spliceozomy genetika MeSH
- steroidy metabolismus MeSH
- vývojová regulace genové exprese genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ecd protein, Drosophila MeSH Prohlížeč
- ekdyson MeSH
- prekurzory RNA MeSH
- proteiny Drosophily MeSH
- ribonukleoproteiny malé jaderné MeSH
- steroidy MeSH
The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- chromatografie iontoměničová MeSH
- chromatografie kapalinová MeSH
- exocytóza genetika fyziologie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hypokotyl genetika růst a vývoj metabolismus MeSH
- proteiny huseníčku genetika metabolismus fyziologie MeSH
- pyl genetika růst a vývoj metabolismus MeSH
- pylová láčka genetika růst a vývoj metabolismus MeSH
- rostlinné proteiny genetika metabolismus fyziologie MeSH
- semenáček genetika růst a vývoj metabolismus MeSH
- tabák genetika růst a vývoj metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- techniky dvojhybridového systému MeSH
- vazba proteinů MeSH
- vezikulární transportní proteiny genetika metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- EXO70A1 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- rostlinné proteiny MeSH
- SEC8 protein, Arabidopsis MeSH Prohlížeč
- vezikulární transportní proteiny MeSH
