A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways

Abstract

Myotonic dystrophy type 2 (DM2) is a genetically defined disease caused by a toxic expanded repeat of r(CCUG) [r(CCUG)^exp], harbored in intron 1 of CCHC-type zinc-finger nucleic acid binding protein (CNBP) pre-mRNA. This r(CCUG)^exp causes toxicity via a gain-of-function mechanism, resulting in three pathological hallmarks: aggregation into nuclear foci; sequestration of muscleblind-like-1 (MBNL1) protein, leading to splicing defects; and retention of CNBP intron 1. We studied two types of small molecules with different modes of action, ones that simply bind and ones that are templated by r(CCUG)^exp in cells, i.e., the RNA synthesizes its own drug. Indeed, our studies completed in DM2 patient-derived fibroblasts showed that the compounds disrupt the r(CCUG)^exp-MBNL1 complex, reduce intron retention, subjecting the liberated intronic r(CCUG)^exp to native decay pathways, and rescue other DM2-associated cellular defects. Importantly, this study shows that small molecules can modulate RNA biology by shunting toxic transcripts toward native decay pathways.

Keywords: RNA; chemical biology; click chemistry; drug design; intron retention; medicinal chemistry; microsatellite disease; myotonic dystrophy; nucleic acids; repeat expansion disorder.

Figure 1:. The RNA that causes DM2, r(CCUG)^exp, has multiple modes of toxicity.

(A) r(CCUG)^exp in intron 1 of CNBP folds into a hairpin displaying a periodic array of internal loops that sequester MBNL1. Sequestration of MBNL1 by r(CCUG)^exp causes (B) pre-mRNA splicing defects, for example increasing IR exon 11 exclusion and (C) aberrant retention of CNBP intron 1. (D) MBNL1 knock-down using siRNA and its effect on relative abundance of MBNL1, CNBP intron 1, and CNBP mature mRNA as measured by RT-qPCR. (E) MBNL1 knock-in using a transfected MBNL1 plasmid and its effect on relative abundance of MBNL1, CNBP intron 1, and CNBP mature mRNA as measured by RT-qPCR. Error bars represent SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by a one-way ANOVA by comparison to untreated cells (“0”; n = 3). See also Figure S1.

Figure 2:. Design and biological impact of small molecules targeting r(CCUG)^exp.

(A) Structures of a kanamycin derivative (green spheres) that avidly binds the internal loops found in r(CCUG)^exp and a dimer composed of two kanamycin binding modules connected via a propylamine peptoid (1). (B) Schematic of 1’s mode of action, binding to r(CCUG)^exp and releasing MBNL1. (C) Structures of compounds employed in an on-site drug synthesis approach. Compound 2, a modified kanamycin module, contains alkyne and azide moieties that react upon binding r(CCUG)^exp, the catalyst. Compound 3 is a kanamycin module containing only the alkyne component and thus cannot oligomerize. (D) Schematic of on-site click synthesis of 2, catalyzed by binding to r(CCUG)^exp, and release MBNL1 to relieve DM2-associated defects.

Figure 3:. Biological activity of 1 in DM2 patient-derived fibroblasts.

(A) Representative microscopic images for the reduction of nuclear foci by 1, as completed by RNA-FISH and immunohistochemistry using an anti-MBNL1 antibody. (B) Quantification of r(CCUG)^exp-MBNL1 foci/nucleus (n = 3 biological replicates, 40 nuclei counted per replicate). (C) Effect of 1 on IR pre-mRNA splicing regulated by MBNL1. (D) Effect of 1 on CNBP pre-mRNA splicing. (E) Effect of 1 on an MBNL1-regulated alternative splicing event, IR exon 11, as determined by RT-PCR; (F) Effect of 1 on aberrant CNBP pre-mRNA splicing caused by r(CCUG)^exp, as determined by RT-PCR; that is, retention of intron 1; (G) Effect of 1 on total intron 1 levels and CNBP mature mRNA, as determined by RT-qPCR. Error bars represent SD. *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by a one-way ANOVA (n = 3). See also Figure S2.

Figure 4:. Biological activity of 2 in DM2 patient-derived fibroxblasts.

(A) Representative microscopic images for the reduction of nuclear foci by 2, as completed by RNA-FISH and immunohistochemistry using an anti-MBNL1 antibody. (B) Quantification of r(CCUG)^exp-MBNL1 foci/nucleus (n = 3 biological replicates, 40 nuclei counted per replicate). (C) Effect of 2 on IR pre-mRNA splicing regulated by MBNL1. (D) Effect of 2 on CNBP pre-mRNA splicing. (E) Effect of 2 on an MBNL1- regulated alternative splicing event, IR exon 11, as determined by RT-PCR; (F) Effect of 2 on aberrant CNBP pre-mRNA splicing caused by r(CCUG)^exp, as determined by RT-PCR; that is, retention of intron 1; (G) Effect of 2 on total intron 1 levels and CNBP mature mRNA, as determined by RT-qPCR. Error bars represent SD. *P < 0.5, **P < 0.01, ***P < 0.001, as determined by a one-way ANOVA (n = 3). See also Figure S3.