Identification of novel protein interactions in the eIF4B-RNA complex by tandem affinity purification using a chimeric construct of His-eIF4B-Flag

摘要

Our attempt to discover novel eIF4B-interacting proteins by mass spectrometry approach led to the identification of nucleolar RNA helicase DDX21 The latter protein as well as many other components of pre-ribosomal particle complexes has been independently found associated with eIF4B by another group. Confocal microscopy has shown partial co-localization of tagged eIF4B and DDX21 in nucleolar periphery. Pulse chase experiments metabolically labeling rRNA show an attenuated 28S rRNA production and concomitant accumulation of 36S intermediates in eIF4B-silenced cells. Since ribosomal biogenesis is highly coordinated process and requires strict stoichiometry maintenance of ribosomal components the observed inhibition of rRNA processing could be consequential to the decreased ribosomal protein expression.