Figure 3. The RING domain of ZNF598 is essential for ribosome stalling at polyA residing within coding sequences.

(a) Schematic diagram of the reporter constructs sandwiching a polybasic oligopeptide track between the fluorescent GFP and mCherry (mCh) fusion protein. (ACT AGC)₆ [(ThrSer)₆] encoded a neutrally charged amino-acid tract that served as a control. (b) Detection of GFP and mCherry fluorescent signals by FACS analyses in samples from (a) shown as relative cell numbers. Each experiment was performed in triplicates. (c) Domain structures of ZNF598 full-length and truncation mutants, with numbers referring to the position of amino acids. (d) Detection of GFP and mCherry fluorescent signals by FACS analyses in samples expressing full-length or truncated versions of ZNF598 and transiently transfected with GFP-mCherry reporter with (ACT AGC)₆ and (AAA)₁₂ linkers. Each experiment was performed in triplicate. (e) Upper panel: autoradiograph of cross-linked, ³²P-labelled, RNA- Flag/HA-ZNF598 immunoprecipitate. Flag/HA-tagged full-length ZNF598 or truncated versions were separated by SDS-PAGE after 4SU PAR-CLIP. Lower panel: Anti-HA Western blot analyses of the cross-linked RNA-protein immunoprecipitates; HC, antibody heavy chain.