Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human

Abstract

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Graphical Abstract

Figure 1.

UUU codon repeats could trigger robust ribosomal frameshifting. (A) Schematic diagram of the P2A dual luciferase system (RLuc, Renilla luciferase; FLuc, firefly luciferase). 0F, +1F or −1F FLuc indicate the FLuc sequence is placed in the 0, +1 or −1 frame, respectively. 0, +1 and −1 UAA mean that a UAA stop codon was added in the 0, +1 or −1 frame, respectively. (B) Bar plot showing frameshifting ratio of HIV −1 PRF sequence in HEK293T cells by luciferase assays. SSm, slippery site mutation (UUUUUUA to CUUCUUA). SSd, slippery site deletion. (C) Bar plot showing Renilla luciferase (left) and firefly luciferase (right) activity of OAZ1 sequences. +1F and 0F means construct with FLuc in the +1 or 0 frame, as shown in (A) The cartoon on top showed the reporter structure. (D) Detection of ribosomal frameshifting induced by different number of UUU codon repeats with dual luciferase systems (top panel) and western blots (bottom panel). EV, empty vector. (E) Frameshifting ratio induced by (UUU)4 or (UUU)5 insertion (INS) with different vectors shown in (A). +1FLuc and −1FLuc means construct with FLuc in the +1 or −1 frame. (F) Schematic diagram of Stop-Go (SG) system. SG1 and SG2 indicate two F2A sequence. (G) Frameshifting ratios (top) and western blot results (bottom) from SG system with different number of UUU repeat insertion in HEK293T cells. EV, empty vector. (H) Schematic diagram of constructs (mC-E system) with OAZ1 (top) and (UUU)5 (bottom) insertion. 0, +1 and −1 mean the position of first stop codon in the corresponding frame after mCherry coding sequence. (I) Western blot analysis for the frameshifting proteins induced by OAZ1 sequence. NV, transfection without vector. (J) Western blot analysis the frameshifting induced by (UUU)5 codon in HEK293T cells. In this figure, FS stands for ribosomal frameshifting, EV for empty vector, error bars for standard deviation of three biological replicates. (*) P< 0.05, (**) P < 0.01 (Student's t-test for 2 data sets and one-way ANOVA for >2 data sets).

Figure 2.

Codon repeat-induced ribosomal frameshifting may widely occur in humans. (A) Frameshifting ratio determined by the P2A dual luciferase system from different types of codon repeat (5-time repeats) in HEK293T cells. Error bars, standard deviation of 3 biological repeats. (B) Detection of ribosomal frameshifting of (UGG)5 in the SPIDR gene. Diagram (top) shown the structure of the construct, with the dark green box indicating the position of (UGG)5 repeats. Middle and bottom panel showing the western blot result and luciferase activity assay. Three biological replicates were analyzed for each experiment. Error bars, standard deviation. (**) P < 0.01 (Student's t-test). (C) Diagram illustrating the definition of CRFS locus, trans-frame peptide and out-of-frame peptide in the trans-frame protein. Red and black asterisk mean the position of first stop codon in the +1 and 0 frame, respectively. (D) Pie chart showing the distribution of peptides detected in human tissue proteomes. Trans-frame or out-of-frame were defined in (C). Annotated protein means peptide sequences match unexpressed annotated proteins. Small peptide means sequences match the smProt database. Splice site means trans-frame peptides positioned at splicing junctions. (E) Venn diagram showing the number of CRFS loci supported by trans-frame, out-of-frame peptides or both. (F) Venn diagram showing the number of CRFS loci supported by trans-frame peptides detected in proteomic data of each biological repeats. (G) Diagram showing the number of CRFS loci supported by unique trans-frame and/or out-of-frame peptides from proteomic data of 32 human tissues. (H) Unique trans-frame peptides detected in two biological repeats and represented in > 16 human tissues. Red sequences are encoded by +1 frame, blue by −1 frame, and black by 0 frame.

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.

Figure 4.

HDAC1-FS affects histone acetylation, cell migration and apoptosis. (A) Schematic diagrams of mCherry or EGFP constructs used to detect the protein localization. (B) Confocal microscopy showing subcellular localization of the HDAC1-FS and HDAC1 protein in HeLa cells with constructs shown in (A). (C) Metagene analyses of H3K9ac ChIP-seq with ectopic expression of 3× Flag-tagged HDAC1-FS, HDAC1 or empty vector (EV). (D) Wound closure assay showing the migration of A549 cells with ectopic expression of Flag-HDAC1-FS (left) and siRNA against HDAC1 (right). EV, empty vector. Error bars, standard deviation of three biological repeats. (**) P < 0.01 (one-way ANOVA). (E) Transwell assay showing the migration of A549 cells with ectopic expression of Flag-HDAC1-FS (left) and with siRNA against HDAC1 (right). EV, empty vector. Error bars, standard deviation of 3 biological repeats. (**) P < 0.01 (Student's t-test for 2 data sets and one-way ANOVA for >2 data sets). (F) Bar plot showing the changes of ribosomal frameshifting (FS) with thapsigargin (TG) or DMSO treatment of in HEK293T cells. The frameshifting ratios determined by P2A luciferase system. Error bars, standard deviation of 3 biological repeats. (**) P < 0.01(Student's t-test). (G) Apoptosis level measured by Annexin V-FITC/PI assay in thapsigargin (TG) treatment HEK293T cells. Three biological replicates were analyzed for each experiment in HEK293T cells. Error bars represent standard deviation. (**) P <0.01(Student's t-test). (H) Schematic diagram showing the experimental design for evaluating the effect of HDAC1-FS in cell apoptosis. (I) Representative western blot result detects HDAC1 protein after transformed with different constructs in wild-type (WT) HEK293T or HDAC1 KO HEK293T cells. EV, empty vector. NV, no transfection. (J) Flow cytometry analyses to TG-induced apoptosis in HEK293T cells transformed HDAC1(wt) and HDAC1(sm). Results are normalized with empty vector (EV). Error bars, standard deviation of three biological repeats. (**) P < 0.01 (Student's t-test).