Neuronal depolarization and CaM kinase IV signaling alter the splicing of multiple exons in transcripts for ion channels, neurotransmitter receptors, and other synaptic proteins. These splicing changes are mediated in part by special CaM kinase-responsive RNA elements, within or adjacent to exons that are repressed in the initial phase of chronic depolarization. The splicing of many neuronal transcripts is also regulated by members of the Fox (Feminizing gene on X) protein family, and these Fox targets are also often proteins affecting synaptic activity. We show that Fox-1/Ataxin 2-Binding Protein 1 (A2BP1), a protein implicated in a variety of neurological diseases, can counteract the effects of chronic depolarization on splicing. We find that exon 19 of Fox-1 is itself repressed by depolarization. Fox-1 transcripts missing exon 19 encode a nuclear isoform of Fox-1 that progressively replaces the cytoplasmic Fox-1 isoform as cells are maintained depolarizing media. The resulting increase in nuclear Fox-1 leads to the reactivation of many Fox-1 target exons, including exon 5 of the NMDA receptor 1, that were initially repressed by the high-KCl medium. These results reveal a novel mechanism for the slow modulation of splicing as cells adapt to chronic stimuli: The subcellular localization of a splicing regulator is controlled through its own alternative splicing. Alternative pre-mRNA splicing is an important mechanism for controlling gene expression in metazoan organisms. Changes in splice site or exon usage frequently determine the function of a gene product or eliminate its expression (Black 2003;Matlin et al. 2005;Blencowe 2006). The best understood systems of splicing regulation are those modulated by cell type, where stable differences in pre-mRNA-binding protein expression determine a splicing choice. However, splicing is also dynamically regulated by external stimuli and growth conditions. Most cellular signaling pathways that alter gene transcription also alter groups of alternative exons (Shin and Manley 2004;Blaustein et al. 2007;Stamm 2008). In some cases, these responses have been traced to particular regulatory elements in the pre-mRNA, to post-translational modifications of splicing regulators, or to changes in transcriptional control. However, the mechanisms that allow the splicing machinery to respond to dynamic stimuli are not well understood.A particularly interesting regulatory mechanism in excitable cells is their response to membrane depolarization. Depolarization-induced changes in neuronal transcription are well studied and involve the activation of CaM kinase and other signaling pathways to induce transcription of both rapid primary response genes and more slowly developing secondary responses (Flavell and Greenberg 2008;Greer and Greenberg 2008). These transcriptional programs play important roles in neuronal plasticity and modulating synaptic activation. Ion channel mRNAs and other transcripts affecting membrane activity also often contain exons whose splicing responds to chronic depolar...

Transcriptomes provide a myriad of potential RNAs that could be the targets of therapeutics or chemical genetic probes of function. Cell permeable small molecules, however, generally do not exploit these targets, owing to the difficulty in the design of high affinity, specific small molecules targeting RNA. As part of a general program to study RNA function using small molecules, we designed bioactive, modularly assembled small molecules that target the non-coding expanded RNA repeat that causes myotonic dystrophy type 1 (DM1), r(CUG)exp. Herein, we present a rigorous study to elucidate features in modularly assembled compounds that afford bioactivity. Different modular assembly scaffolds were investigated including polyamines, α-peptides, β-peptides, and peptide tertiary amides (PTAs). Based on activity as assessed by improvement of DM1-associated defects, stability against proteases, cellular permeability, and toxicity, we discovered that constrained backbones, namely PTAs, are optimal. Notably, we determined that r(CUG)exp is the target of the optimal PTA in cellular models and that the optimal PTA improves DM1-associated defects in a mouse model. Biophysical analyses were employed to investigate potential sources of bioactivity. These investigations show that modularly assembled compounds have increased residence times on their targets and faster on rates than the RNA-binding modules from which they were derived and faster on rates than the protein that binds r(CUG)exp, the inactivation of which gives rise to DM1-associated defects. These studies provide information about features of small molecules that are programmable for targeting RNA, allowing for the facile optimization of therapeutics or chemical probes against other cellular RNA targets.

Biomarker-driven trials hold promise for therapeutic development in chronic diseases, such as muscular dystrophy. Myotonic dystrophy type 1 (DM1) involves RNA toxicity, where transcripts containing expanded CUG-repeats (CUGexp) accumulate in nuclear foci and sequester splicing factors in the Muscleblind-like (Mbnl) family. Oligonucleotide therapies to mitigate RNA toxicity have emerged but reliable measures of target engagement are needed. Here we examined muscle transcriptomes in mouse models of DM1 and found that CUGexp expression or Mbnl gene deletion cause similar dysregulation of alternative splicing. We selected 35 dysregulated exons for further study by targeted RNA sequencing. Across a spectrum of mouse models, the individual splice events and a composite index derived from all events showed a graded response to decrements of Mbnl or increments of CUGexp. Antisense oligonucleotides caused prompt reduction of CUGexp RNA and parallel correction of the splicing index, followed by subsequent elimination of myotonia. These results suggest that targeted splice sequencing may provide a sensitive and reliable way to assess therapeutic impact in DM1.

Significance statement. Many different diseases are caused by toxic structured RNAs.Herein, we designed a lead small molecule that binds a toxic structure and rescues disease biology. We show that a structure-specific small molecule can improve disease-associated defects in two diseases that share the common toxic RNA structure. In one disease, the toxic structure is harbored in an intron and causes its retention. The compound facilitates processing of a retained intron, enabling the disease-affected cell to remove the toxic RNA.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients’ cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.