Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the β1-β2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.
- MeSH
- ADP-ribosylační faktor 1 chemie genetika MeSH
- cytoplazma chemie genetika MeSH
- cytoskeletální proteiny chemie genetika MeSH
- dvouvláknová RNA chemie genetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- proteiny vázající RNA chemie genetika MeSH
- stabilita RNA genetika MeSH
- vazebná místa genetika MeSH
- vazebný motiv pro dvoušroubovici RNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many plant viruses are vectored by insects in a persistent circulative manner. The insect gut and salivary gland are important barriers limiting virus spread, but the mechanisms by which viruses are able to cross the gut escape barriers of the insect remain largely unknown. Wheat dwarf virus (WDV), transmitted by Psammotettix alienus in a persistent, circulative, and nonpropagative manner, causes the most economically important virus disease in wheat. In this study, ADP ribosylation factor 1 (ARF1) was found to interact with the coat protein of WDV in a yeast two-hybrid, pull-down assay and to colocalise with virions in the gut and salivary glands of P. alienus. When transcription of ARF1 was suppressed by RNA interference, the WDV titre decreased in the haemolymph and salivary glands, and transmission efficiency decreased, but titre in the gut did not differ from that of the control. These data suggest that ARF1 of P. alienus binds to the WDV virion and helps virus spread from gut to haemolymph. Our study provides direct experimental evidence that WDV can use the existing membrane trafficking mechanism to aid its spread within the insect vector. This first analysis of the molecular interaction between WDV and its vector P. alienus contributes to understanding the mechanisms involved in circulative transmission of the virus by the leafhopper vector.
- MeSH
- ADP-ribosylační faktor 1 genetika metabolismus MeSH
- buněčné linie MeSH
- Geminiviridae patogenita MeSH
- Hemiptera genetika metabolismus virologie MeSH
- hmyz - vektory genetika MeSH
- nemoci rostlin virologie MeSH
- RNA interference MeSH
- slinné žlázy metabolismus virologie MeSH
- střeva virologie MeSH
- techniky dvojhybridového systému MeSH
- virion metabolismus MeSH
- virové plášťové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
- MeSH
- ADP-ribosylační faktor 1 genetika metabolismus MeSH
- buněčná membrána enzymologie MeSH
- endozomy enzymologie MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- genové regulační sítě MeSH
- HeLa buňky MeSH
- lidé MeSH
- mapy interakcí proteinů MeSH
- rac1 protein vázající GTP genetika metabolismus MeSH
- receptor IGF typ 2 genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- RNA interference * MeSH
- shluková analýza MeSH
- signální transdukce MeSH
- skupina kinas odvozených od src-genu genetika metabolismus MeSH
- trans-Golgiho síť enzymologie MeSH
- transfekce MeSH
- transport proteinů genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
