Analysis of ALKBH8-bound tRNAs identified by HITS-CLIP. (A) List of tRNAs identified by HITS-CLIP. The displayed tRNAs are the top-scoring tRNAs according to the CPM in Supplemental Table S2. The tRNAs also observed by ALKBH8 RIP-seq analysis are highlighted in gray. The first column (top left) are the previously known ALKBH8 tRNA targets. tRNAs are displayed according to the identity of the wobble nucleotide. (B) Northern blot analysis of selected tRNAs coprecipitated with ALKBH8. Empty are HEK293T cells without the integrated FLAG-ALKBH8 gene. ALKBH8 are stable cell lines that express FLAG-ALKBH8. Input is RNA isolated from whole cell extracts. ALKBH8 IP are RNAs coprecipitated with affinity-purified ALKBH8. Beads lacking anti-FLAG antibody were used as a control for nonspecific binding of tRNA (−). FLAG-coupled beads are marked as +. DNA oligo probes for individual tRNAs were 5′ terminal labeled by γ32P-ATP. The RNAs were resolved on 20% polyacrylamide gel and the radioactive signals detected by autoradiography. (C) Heatmap representation of ALKBH8 binding to tRNAs as revealed by the HITS-CLIP analysis. The strong signal of tRNA-Gly-TCC was omitted in order to decrease the scale of the heat plot. All gene lengths were scaled to an equal length of 74 nt on the x-axis. The color scale on the right represents the CPM normalized number of mapped reads to a region. (D) Schematic representation of ALKBH8 binding to the secondary structure of tRNA. The areas with the highest mapping are highlighted by a thick black line, and the lower number of mapped reads is marked in thick gray. (E) Proportions of CPM normalized values of nontemplated 3′-terminal extensions in most represented tRNAs detected by RIP-seq. The red bars include all the different versions of 3′ extensions (C, CC, CCA, see details in Supplemental Fig. S1D). The number of tRNA reads without any nontemplated 3′-terminal extensions are in gray.
