Fig. 1: Hakai interacts with MACOM components.
From: Hakai is required for stabilization of core components of the m6A mRNA methylation machinery
a Table indicating the names of MAC and MACOM components in Vertebrates and Drosophila. b Schematic of the Hakai short and long protein isoforms depicting the RING domain (pink), the Zn-finger (grey) and the HYB domain (black). The green domain indicates the alternative region of the second exon that is included in isoforms 311 aa and 473 aa. c Immunostaining of Myc-tagged Hakai short and long protein isoforms overexpressed in S2R+ cells. GFP-tagged Barentsz protein served as a cytoplasmic marker. DAPI staining is shown in blue. The short Hakai isoform localizes strictly to the cytoplasm, whereas the long isoform localizes to both cellular compartments with the enrichment in the nucleus. Scale bars, 10 μm. d Identification of proteins interacting with GFP-Hakai-long in S2R+ cells based on label-free analysis of two replicate experiments analyzed by quantitative MS-based proteomics. HAKAI immunoprecipitates alongside of all MACOM components. MACOM component proteins are highlighted in red. A complete list of quantified proteins can be found in Supplementary Data 2. e, f Co-immunoprecipitation experiments were carried out with lysates prepared from S2R+ cells transfected with Myc-tagged Hakai (long isoform) and HA-tagged Nito or Fl(2)d. In control lanes, S2R+ cells were transfected with Myc alone and an identical HA-containing protein. Extracts were immunoprecipitated with anti-Myc antibody and immunoblotted using anti-Myc and anti-HA antibodies. Two percent of input was loaded. The same experiment was repeated in the presence of RNase T1. Nito and Fl(2)d interact with Hakai in an RNase-independent manner. Blots shown are representative of one biological replicate. g Yeast-two-hybrid assay to investigate Hakai interaction with Nito and Fl(2)d. Proteins were cloned in yeast expression vectors and fused with either Gal4-DNA binding domain (BD) or Gal4-DNA activation domain (AD). Indicated combinations of vectors were co-expressed in yeast and empty vectors encoding only activation or binding domain were used as control (Ctr). Recovered colonies were spotted on plates lacking Leucine and Tryptophan (-Leu, -Trp) as well as selection plates lacking amino acids Leucine, Tryptophan and Histidine (-Leu, -Trp, -His). AD-Hakai long isoform interacts with BD-Fl(2)d and BD-Nito. Source data for Western blots and the yeast-two-hybrid assay are provided as a Source Data file.
