Determination of Dicer-TRBP interaction domains and colocalization. A) Dicer interacting domain is located in a unique region between the ATPase and the helicase domains. (Top) Schematic representation of Dicer protein and its domains. (Bottom) Dicer 173–431 or 267–541 represent the isolated interacting Dicer clones in a two-hybrid screen using TRBP as bait. Yeast two hybrid assay using Dicer or Dicer domains expressed in pGADGH vector and their interactions with TRBP2 clones expressed in pGBT9 vector. B) The Medipal subdomain in TRBP is located between aa 298 and 366. (Top) Schematic representation of TRBP2 protein and its domains. (Bottom) Yeast two hybrid assay using TRBP2 or TRBP domains expressed in pGADGH vector and their interactions with Dicer domains identified in A and expressed in pGBT9 vector. NG means no growth. Shown are representative assays of three independent experiments. Dicer and TRBP2 schematic representation of full-length proteins are at scale. C) Endogenous TRBP and Dicer colocalize. HeLa cells were fixed and labeled with rabbit anti-TRBP673 and mouse anti-Dicer (mAb42) followed by anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 546. The cells were mounted and assayed for fluorescence by laser scanning confocal microscopy on a Zeiss LSM Pascal 5 microscope. Alexa Fluor 488 and Alexa Fluor 546 fluorescences are represented as green and red, respectively, while merged colors appear as yellow. Images showing the colocalization of the two proteins detected by the yellow dots are amplified on the right.
Daniels et al. BMC Molecular Biology 2009 10:38 doi:10.1186/1471-2199-10-38