The Dbp5 cycle at the nuclear pore complex during mRNA export I: Dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Christine A. Hodge, Elizabeth J. Tran, Kristen N. Noble, Abel R. Alcazar-Roman, Rakefet Ben-Yishay, John J. Scarcelli, Andrew W. Folkmann, Yaron Shav-Tal, Susan R. Wente, Charles N. Cole

Research output: Contribution to journalArticlepeer-review

Abstract

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP6). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP6/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.

Original languageEnglish (US)
Pages (from-to)1052-1064
Number of pages13
JournalGenes and Development
Volume25
Issue number10
DOIs
StatePublished - May 15 2011
Externally publishedYes

Keywords

  • DEAD-box proteins
  • Dominant-negative mutants
  • FRAP
  • Nuclear pore complex
  • Nucleocytoplasmic transport

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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