Fig. 4.

iCLIP identifies the ARPP21–RNA interactome. a ARPP21 deletion constructs used in UV-crosslinking experiments to assess their RNA-binding potential. Construct 2 conforms to a splice variant that lacks a part of the SUZ domain. b SDS-PAGE with FLAG immunoblot confirms immunoprecipitation of all ARPP21 variants, lane designations refer to constructs depicted in a. c Autoradiogramm of crosslinked and ³²P-labeled ARPP21–RNA complexes separated by NuPAGE, lane designations as in a and b. All variants harboring at least one of the predicted RNA-binding domains exhibit RNA-binding ability (*), the C-terminal only Construct 6 yielded no signal. d Schematic overview of the iCLIP procedure performed with iARPP21 cells. Unique barcodes (blue) introduced during the reverse transcription allow for multiplexing iCLIP samples. Due to the circularization step, truncated cDNAs are captured and the position of protein–RNA crosslinking (red) can be accurately assessed. Green: deep-sequencing adapters. e Representative ARPP21 iCLIP coverage at the Phf6 locus shows specificity for the 3′UTR. The negative control iCLIP from cells not expressing ARPP21 shows negligible background. Numerical scale on left refers to unique crosslinking events (CEs). f Genome-wide characterization of unique CEs for ARPP21 by RNA type. g 3′UTRs of ARPP21 targets are longer compared to non-ARPP21 target transcripts. Median 3′UTR length of ARPP21 target mRNA: 1.99 kb. Median 3′UTR length of ARPP21-unbound mRNA: 0.72 kb. h Nucleotide representation flanking the crosslink site as determined by GraphProt identifies a uridine-rich motif. i ARPP21 iCLIP signals are enriched at uridine-rich sequences. A representative peak from the Phf6 3′UTR is shown; the numerical scale at left refers to unique CEs. j Secondary structure analysis of the ARPP21 interaction motif by GraphProt. The center of the motif containing the crosslinked nucleotide is largely devoid of predicted stems. E external loop, H hairpin, I internal loop, M multi loop, S stem