Fig 1: The ribosome-binding activity of yeast Lso2 is conserved in its human ortholog.(A) (Top) Schematic of coiled-coil domains in Lso2 and CCDC124. (Bottom) Protein sequence alignment of yeast Lso2 with human CCDC124. (B) HeLa cell extract was fractionated through a sucrose gradient containing 5 mM magnesium. Equivalent fraction volumes were TCA precipitated and loaded in each lane. Fractions were probed for RPS5 and for endogenous CCDC124. (C) For both strains, cell extracts were fractionated through a sucrose gradient and the fractions probed for Asc1 and V5. (Top) The yeast LSO2 gene was tagged with V5 in a marker-free insertion. (Bottom) The yeast LSO2 gene was swapped with V5-tagged CCDC124 in a marker-free replacement. (D) (Left) Internally normalized read coverage across the RDN37 locus in CCDC124-Myc ePAR-CLIP libraries. The y-axis is reads per million reads mapping to RDN37. The blue region is the 25S rRNA cluster that reproducibly crosslinked to Lso2-Myc (Fig 2B). (Right) Inset of RDN37-normalized read coverage across the RDN25 region crosslinking to Lso2-Myc. (E) Normalized read coverage across a series of negative-strand tRNA features in CCDC124-Myc ePAR-CLIP libraries. Y-axis is reads per million reads in the entire library. See also S3 Fig and S3 Table. CCDC124, coiled-coil domain containing 124; ePAR-CLIP, photoactivatable ribonucleoside crosslinking and immunoprecipitation and an enhanced method of CLIP library preparation; IP, immunoprecipitation; Lso2, late-annotated short open reading frame 2; Lso2-Myc, yeast strains expression Myc-tagged Lso2 at endogenous levels; RPS5, ribosomal protein S5; TCA, trichloroacetic acid.