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

Abstract

2 HIGHLIGHTS• Intron retention in RNA repeat expansions can be due to repeats binding to proteins • Small molecules that bind RNA repeats and inhibit protein binding can trigger decay • A toxic RNA repeat can catalyze the synthesis of its own inhibitor on-site• On-site drug synthesis most potently affects disease biology eTOC BLURBThe most common way to target RNA is to use antisense oligonucleotides to target unstructured RNAs for destruction. Here, we show for the first time that small molecules targeting structured, disease-causing RNAs can shunt them towards native decay pathways by affecting their processing. SUMMARYMyotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy caused by a toxic expanded repeat of r(CCUG) [heretofore (CCUG) exp ], harbored in intron 1 of CHC-Type Zinc Finger Nucleic Acid Binding Protein (CNBP) pre-mRNA. This r(CCUG) exp causes DM2 via a gain-offunction mechanism that results in three hallmarks of its pathology: (i) binding to RNA-binding proteins (RBPs) that aggregate into nuclear foci; (ii) sequestration of muscleblind-like-1 (MBNL1) protein, a regulator of alternative pre-mRNA splicing, leading to splicing defects; and (iii) retention of intron 1 in the CNBP mRNA. Here, we find that CNBP intron retention is caused by the r(CCUG) exp -MBNL1 complex and can be rescued by small molecules. We studied two types of small molecules with different modes of action, ones that simply bind and ones that can be synthesized by a r(CCUG) exp -templated reaction in cells, that is the RNA synthesizes its own drug.Indeed, our studies completed in DM2 patient-derived fibroblasts show 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.Collectively, this study shows that small molecules can affect RNA biology by shunting toxic transcripts towards native decay pathways. processing will be required to understand and capture the full potential of compounds to target RNA and rescue disease biology. The development of chemical probes and lead medicines targeting structured RNA, particularly human RNAs, are in their infancy. It will be interesting to see the types of chemical structures, their features, and the functional responses that emerge.

Structure-Specific Cleavage of an RNA Repeat Expansion with a Dimeric Small Molecule Is Advantageous over Sequence-Specific Recognition by an Oligonucleotide

“…In our previous studies, we developed a potent dimer ( 1 ) that selectively targeted r­(CCUG) exp and triggered native decay of the intron 1 species (Figure A). , Dimer 1 rescued IR pre-mRNA splicing defects and, importantly, does not affect IR splicing in healthy cells (Figures S1 and S2). Additionally, no effect was observed on the inclusion of mitogen-activated protein kinase kinase kinase kinase 4 ( MAP4K4 ) exon 22a, a non-MBNL1 dependent splicing event (Figure S3).…”

“…Although the designer binding compound liberated the retained intron, thereby subjecting it to decay, the percentage eliminated was only ∼20%; notably, levels of CNBP mature mRNA were unaffected (Figure S4). Thus, to eliminate this transcript to a greater extent and more potently improve DM2 defects, we designed a version of the binding compound 1 that would directly subject the transcript to decay by appending it to bleomycin A5 ( 2 , Figure A). Attachment of bleomycin A5 to an RNA-binding module using its free amine affords a compound that is capable of selective cleavage of an RNA target while ablating DNA cleavage at the active concentration, , as the free amine forms key interactions that contribute to DNA affinity (Figure B) .…”

“…Among other defects, r­(CCUG) exp causes aberrant intron retention. The small molecule liberates the retained intron, which is subsequently decayed . Herein, we describe the development of a small molecule that directly cleaves r­(CCUG) exp and rescues disease-causing defects.…”

A small molecule that binds an RNA repeat expansion stimulates its decay via the exosome complex

“…It is very likely that specific targeting of RNA structure can facilitate the recognition of RNA by the quality control machinery by using very simple ligands such as 2b. Furthermore, the observed selectivity of a small molecule could be even greater than expected if off-target binding occurs in a non-functional site that has no effect on downstream biology (39 (12) including as DM2 (29) and c9ALS/FTD (40), and as such it will be interesting to see if the same pathway can be activated with ligands that target these repeat expansions. If so, these targets may be more druggable than they immediately appear.…”

“…In myotonic dystrophy type 2 (DM2), intron retention is due, at least in part, to formation of the r(CCUG) exp -MBNL1 complex (29). Inhibition of this complex via small molecule treatment triggered decay of the retained intron, but there was no mechanistic basis for this observation, for example which enzymes were responsible for the intron's degradation (29). To study the levels of TCF4 in FECD cells, we performed RT-qPCR using primers for mature TCF4 mRNA and primers specific for r(CUG) exp -containing intron 3.…”