Identification of poly(A)+ RNA-bound proteins in fly embryos. (A) Schematic overview of mRNA interactome capture in early yw and X490 Drosophila melanogaster embryos. (B) Oligo(dT) precipitates of UV-crosslinked (+) and noncrosslinked (−) lysed yw and X490 embryos were separated by SDS-PAGE and silver-stained. (C) Scatterplot showing log10-transformed intensities of proteins in oligo(dT) pull-downs from crosslinked embryos versus log10-transformed intensities of proteins in oligo(dT) precipitates from noncrosslinked yw (top) and X490 (bottom) embryos. Proteins with at least two unique peptides and greater than 10-fold intensity in oligo(dT) pull-downs from crosslinked embryos compared to oligo(dT) precipitates from noncrosslinked embryos are considered as enriched and part of the early fly RNA-bound proteome (black). Common contaminant proteins (e.g., trypsin and keratin) are marked in red. (D) Venn diagram depicting the overlap of RNA-bound proteins (n = 1217) identified in yw (n = 1144) and/or X490 (n = 549) embryos. Four hundred seventy-six proteins overlap in both approaches and are referred to as the early fly mRBPome. Seven hundred forty-one proteins were uniquely identified. (E) Overlap of the early fly mRBPome (n = 476) to mRNA interactome studies in mouse mESCs (Kwon et al. 2013) and human HEK 293 and HeLa cells (Baltz et al. 2012; Castello et al. 2012) and (F) to the human RBP census (Gerstberger et al. 2014).
