RNA-PROTACs: Degraders of RNA-Binding Proteins

Abstract

Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs are a new class of drugs that mediate selective degradation of a target protein through a cell's ubiquitination machinery. PROTACs comprise a moiety that binds the selected protein, conjugated to a ligand of an E3 ligase. Herein, we introduce RNA-PROTACs as a new concept in the targeting of RBPs. These chimeric structures employ small RNA mimics as targeting groups that dock the RNA-binding site of the RBP, whereupon a conjugated E3-recruiting peptide derived from the HIF-1α protein directs the RBP for proteasomal degradation. We performed a proof-of-concept demonstration with the degradation of two RBPs-a stem cell factor LIN28 and a splicing factor RBFOX1-and showed their use in cancer cell lines. The RNA-PROTAC approach opens the way to rapid, selective targeting of RBPs in a rational and general fashion.

Keywords: Lin28; PROTAC; RNA-binding proteins; oligonucleotides; proteasomal degradation.

Figure 1

RNA‐PROTACs bind RBPs and direct them for degradation. A) An RNA‐PROTAC comprising a short oligonucleotide binds the RNA‐binding domain of the RBP and mediates its ubiquitination and degradation. B) The Lin28 zinc finger domain binding to its consensus sequence AGGAGAU (adapted from Ref. [13b], PDB: 2LI8).

Figure 2

Fluorescence polarization competition assay with Lin28‐binding RNA mimics. A) ¹H–¹⁵ N HSQC spectra of Lin28_ZKD, free (blue) and bound (red) to ORN1 at a 1:1 ratio. B) Overlay of selected ¹H–¹⁵ N HSQC spectra of Lin28_ZKD, free (blue) and bound (red) to ORN1, ORN2 and ORN3 at 1:1 ratios. C) Sequence of FAM labelled preE‐let‐7f‐1 used in this study; domain structures of Lin28 (ZKD: orange; RNA residues that bind the ZKD: red). D) Fluorescence polarization assay of Lin28_ZKD binding to preE‐let‐7f‐1‐FAM. E) Competition assay using unlabeled preE‐let‐7f‐1. F) Competition fluorescence polarization assays with ORN1, ‐2, ‐3, ‐5 and ‐7. Wild‐type RNA (ORN1) is a weak competitor to preE‐let‐7f‐1. (n=3 replicates) G) The solution structure of Lin28_ZKD bound to 5′‐AGGAGAU‐3. Dotted line identifies possible interaction between Lys150 and the phosphorothioate linkage between residues A₄/G₅ (adapted from Ref. [13b], PDB: 2LI8).

Figure 3

Synthesis of RNA‐PROTACs and their mechanism of action. A) Synthetic route to RNA‐PROTACs and controls. B) Fluorescence polarization assay for ORN3P₁ and negative seq‐control ORN5P₁. C,D) RNA‐PROTAC‐mediated ubiquitination of myc‐Lin28A in 22Rv1 cells co‐transfected with HA‐Ubiquitin (HA‐Ub) and pCMV‐myc‐Lin28A, or pDEST‐Myc‐QKI with either vehicle (OptiMEM) or 500 nM ORN3P₁ (C) or ORN7P₁ (D). cMyc‐immunoprecipitated lysates were separated by SDS‐PAGE followed by western blots detecting HA (Ub). Slow‐migrating smears in right‐hand lanes represent ubiquitin‐conjugated cMyc‐Lin28A. WCL=whole‐cell lysate input.

Figure 4

RNA‐PROTACs mediate Lin28A and RBFOX1 degradation in cells. A) Lin28A and Lin28B localization in K562 cells; Hoechst staining indicates location of nuclei. B) Pull‐down of Lin28A onto streptavidin‐coated microtiter plates from the lysates of K562 cells treated with biotin‐labeled ORN3P1 and ORN7P1. Relative binding is normalized to mock‐treated cells. C) Western blot and quantification of Lin28A from K562 cells incubated with ORN3P₁ and negative seq‐control ORN5P₁ for 48 h. D) RNA‐PROTACs mediate degradation of RBFOX1 in HEK293 cells. HEK293 cells were incubated with ORN9P₁, ORN10P₁ and negative peptide‐control ORN9P_con for 48 h. Data are shown as mean ±SD of three independent experiments for (C) and two for (D). M=cells treated with mock solutions, MG=cells treated with 100 nM MG132, +MG=cells co‐treated with RNA‐PROTACs and 100 nM MG132. Error bars indicate standard deviations. Asterisks denote statistical significance compared to 0 nM dose assessed by 1‐way ANOVA test whereas: ns P>0.05, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001. Uncropped western blots are shown in Figure S14,15.