Extended Data Figure 2. Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1.

a, Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a, presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex^12,14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold²⁴ (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log₂) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61.