Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA

Abstract

Background: Due to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. To further facilitate the application of the PUF domain for the sequence-specific RBP engineering, a rapid cloning approach is desirable that would allow efficient introduction of multiple key amino acid mutations in the protein. Here, we report the implementation of the Golden Gate cloning method for an efficient one-step assembly of a designer PUF domain for RNA specificity engineering.

Results: We created a repeat module library that is potentially capable of generating a PUF domain with any desired specificity. PUF domains with multiple repeat modifications for the recognition of altered RNA targets were obtained in a one-step assembly reaction, which was found to be highly efficient. The new PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular approach for PUF engineering. To demonstrate the application of the PUF domain assembly method for RBP engineering, we fused the PUF domain to a post-transcriptional regulator and observed a sequence-specific reporter and endogenous gene repression in human cell lines.

Conclusions: The Golden Gate based cloning approach thus should allow greater flexibility and speed in implementing the PUF protein scaffold for engineering designer RBPs, and facilitate its use as a tool in basic and applied biology and medicine.

Figure 1

The GG library and assembly schematic. (a) Crystal structure of PUM-HD bound to RNA, adapted from reference [15], GenBank ID code 1M8Y. (b) Schematic of PUM-HD bound to RNA. Filled boxes, PUF modules. Circles, RNA bases. (c) Schematic of the main library components: 8 repeat modules, with matching overhangs colored in identical colors; 4 variations of module 2, with corresponding recognition nucleotide indicated above; the aa sequence of module 2, with mutant aa indicated in red; and two receiving vectors. (d) The GG assembly schematic. A one-pot reaction that contains 8 modules of choice, a receiving vector, and enzymes allows the creation of 9 unique overhangs. The exact matching of the overhangs results in the predetermined repeat order assembled in the receiving vector. (e) Schematic of the GG library. R, module; N, nucleotide; recognized nucleotides indicated in the top row. First and last letters in the module names represent aa residues 12 and 16, in each module, respectively. Middle letter, if present, represents the “stacking” aa 13. Black font, WT modules. Red font, mutant modules. Green, yellow, pink, and blue fillings for modules recognizing A, G, U, and C, respectively.

Figure 2

Various TPUF repression activity assessment in HeLa cell line. (a) Dual luciferase assay shows that TPUF (WT), a fusion of TTP (C147R) and PUM-HD (WT), exhibits the greatest down-regulation activity on FL_{PBS (WT)} expression, compared with TTP and PUM-HD (WT) alone. Data represented as mean fold change relative to cells transfected with FL_Random (dashed line, unrepressed FL/RL activity) ± SD: n.s., not significant, ***P ≤ 0.001 (n = 3, t test). (b) Luciferase assay shows predicted specificity of previously reported PUF mutants [16]. TPUF (WT) prefers PBS (WT), TPUF (1SE) prefers PBS (A8G), and TPUF (6SE,7NQ) prefers PBS (GU/UG). Data represent means ± SD: n.s., not significant, **P ≤ 0.01, ***P ≤ 0.001 (n = 3, t test). (c) Mutations and PBSs of TPUFs A-E with 3-4 randomly chosen mutant modules. Black, WT PUF modules and corresponding RNA bases. Red, mutant PUF modules and corresponding RNA bases. Ct, C-terminus, Nt, N-terminus of the protein. (d) A graph of luciferase activity, where TPUFs A-E repress FLs with cognate PBSs. Data represent means ± SD: **P ≤ 0.01, ***P ≤ 0.001 (n = 3, t test). (e) Western blot of effector proteins using anti-Flag antibody shows no major difference in the expression. Anti-α–tubulin antibody was used as a loading control.

Figure 3

TPUF represses endogenously expressed VEGFA gene in HEK293 cell line. (a) Mutations and binding sequences of TPUFs designed for VEGFA 3′ UTR recognition. Black, WT modules and corresponding RNA bases. Red, mutant modules and corresponding RNA bases. Blue, a mismatch in the recognition sequence. Ct, C-terminus, Nt, N-terminus of the protein. (b) The graph demonstrates inhibition of hypoxia-induced VEGFA expression in cells transfected with engineered TPUFs VEGF3 and VEGF7. In hypoxic (+) cultures, VEGFA expression was induced with 500 μM CoCl₂ 24 hours after transfection and then cultivated for 24 hours. Secreted VEGFA levels measured by ELISA were normalized to total protein amounts from lysed cells measured by Bradford Assay. Data represented as mean ± SD: n.s., not significant, *P ≤ 0.05, **P ≤ 0.01, (n = 3, t test). (c) The graph demonstrates inhibition of DHB-induced VEGFA expression in cells transfected with TPUFs WT, VEGF1, VEGF3, and VEGF7. HEK293 cells with the integrated V24P-GS60 transcriptional activator of endogenous VEGFA promoter were treated with 100 nM DHB 24 hours after TPUF transfection and then cultivated for 24 hours. ELISA and normalization to total protein amounts as in the previous panel. Data represented as mean ± SD: n.s., not significant, *P ≤ 0.05. (d) Western blot of effector proteins using anti-Flag antibody shows greater expression of TPUFs VEGF3 and VEGF7 compared with TPUF (WT) and other mutants. Anti-α–tubulin antibody was used as a loading control.