Figure 1
From: RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific
Schematic for RNAcompete assays and sample experimental data. (a) A GST-tagged RBP (RBP is blue shape, GST-tag is yellow oval) is incubated with a 75-fold excess of a non-random, custom designed RNA pool (multicolored lines). RNA selectively bound to an RBP during a GST-pulldown assay is eluted, directly labeled with either Cy3 or Cy5 (green circles) and hybridized to a custom Agilent 244 K microarray. Microarray data is analyzed computationally to generate RNA-binding motifs, represented as logos. (b) RNAcompete sample data for several classes of ucRBPs—NH2PL1 (“Ribosomal proteins” ucRBP class), ZRANB2 (previously characterized ucRBP class), and LSM6 (“Other” ucRBP class)—represented in Fig. 2. Scatterplots show correlation between 7-mer Z-scores for Set A and Set B sequences. RNAcompete logos derived from the top 10 7-mers from Set A sequences (bottom right corner of scatterplot), Set B sequences (top left corner of scatterplot), and the combined Set A and Set B sequences (top of scatterplot) are indicated. The top three 7-mer sequences are shown in the plots. Figures 1, 2, 3, 4, 5 and 6 were created using Adobe Illustrator version 25.4.1—motifs were made using R (version 4.1.3) with the ggseqlogo package (version 0.1) and scatter plots/histograms were made in R (version 4.1.3) with the ggplot2 package (version 3.3.5, https://ggplot2.tidyverse.org).
