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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
Published on: May 10, 2018
Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA
Wang-Yong Yang1, Henry D Wilson1, Sai Pradeep Velagapudi1
1†Department of Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, United States.
Abstract:
One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.
Insights
New chemical probes target toxic RNA repeats causing Fragile X-associated tremor/ataxia syndrome (FXTAS). These small molecules improve RNA splicing and reduce harmful protein production without impacting normal translation.
Area of Science:
- RNA Biology
- Chemical Biology
- Neurogenetics
Background:
- Expanded repeat RNAs cause disease through mechanisms like protein sequestration and repeat-associated non-AUG (RAN) translation.
- Fragile X-associated tremor/ataxia syndrome (FXTAS) is linked to expanded r(CGG) repeats in the 5' untranslated region of the FMR1 gene.
Purpose of the Study:
- To develop and characterize chemical probes that specifically bind and react with expanded r(CGG) repeats.
- To investigate the therapeutic potential of these chemical probes in correcting RNA-mediated disease pathologies.
Main Methods:
- Chem-CLIP-Map (Chemical Crosslinking, Immunoprecipitation, and Sequencing) was employed to map small molecule binding sites on RNA within cells.
- Assays were performed to evaluate the impact of chemical probes and oligonucleotides on pre-mRNA splicing and RAN translation.
Main Results:
- Chemical probes were shown to bind specifically to expanded r(CGG) repeats in cells.
- The developed compounds effectively improved FXTAS-associated pre-mRNA splicing defects and reduced RAN translation without affecting canonical translation.
- Oligonucleotides targeting the same RNA repeat impaired both RAN and canonical translation by reducing polysome loading.
Conclusions:
- Designer small molecules can serve as effective tools for profiling RNA targets in cells, including identifying binding sites.
- Reactive small molecules offer a potentially more beneficial therapeutic approach than oligonucleotides for modulating RNA-mediated disease pathology, as demonstrated in FXTAS models.
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