Figure 3.

Ribosomal frameshifting efficiently occurs at the (UAC)3 repeat of HDAC1. (A) Cartoon showing constructs for mCherry-HDAC1 (mC-HDAC1) fusion protein. 4–88 nt mean 33 nt plus (UAC)3 repeat and 43 nt downstream of the (UAC)3 repeat. (*), in-frame stop codon. (B) Western blot detecting putative trans-frame proteins. The full length and trans-frame protein were marked by black and red arrows, respectively. (C) Frameshifting ratio of HDAC1 gene was detected by luciferase activity. INS, inserted HDAC1 fragment. 5′ ter and 3′ ter, premature stop at 5′ or 3′ of HDAC1 fragment, details in Figure S4A. (D) Diagrams of the truncated HDAC1 fusion constructs. BGH (Bovine growth hormone), transcription termination sequence. +1 and 0 stop codon mean the end of +1CRFS locus and end of protein, respectively. (E) Western blot detecting the HDAC1-FS trans-frame protein with constructs showed in this Figure (A and D) and Figure S4C. Protein produced by normal translation or +1 frameshifting were marked by black and red arrow, respectively. (F) Schematic diagram of dual fluorescent protein system with HDAC1 insert sequence and two stop codons in the +1 and −1 frame. (G) Flow cytometry analysis of HEK293T cells stably transformed with construct shown in (F). FITC-A and PE-Texas Red-A represent the fluorescence intensity of GFP and mCherry, respectively. (H) Diagram showing the construct for IP-MS and the experimental flow. (I) Table showing detected peptides related to CRFS. Relative positions shown in (H). (J) Diagram showing the frameshifting events at N-terminal 50 codons of HDAC1 from proteomic analyses. Pink and blue triangles represent +1 and −1 frameshifting positions, with number indicating the frequency of detected trans-frame peptides in all 32 tissues. The putative HDAC1-FS sequence resulted from +1CRFS, with representative mass spectrogram of trans-frame peptides is shown in bottom. (K) Heatmap showing the numbers of detected HDAC1 trans-frame peptides in proteomes of each 32 human tissues.