Targeting RNA in mammalian systems with small molecules

Abstract

The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease.

Keywords: G-quadruplex; RNA; RNA in disease; RNA interactions; RNA structure; RNA therapeutic target; biological systems; chemical probes; expanded repeats; ligand; long non-coding RNA; mammalian; microRNA; small molecule; splice sites.

Figure 1

Example cellular processes and interactions regulated by RNA structures in non-coding regions. Left: In the nucleus, structured regions in pre-mRNAs can regulate alternative splicing. Structured non-coding RNAs can recruit transcription factors to genomic loci, interacting with both proteins and DNA. Formation of tRNA-like structures can promote cleavage from a longer transcript and export of the RNA fragment into the cytoplasm. Right: In both the nucleus and the cytoplasm, sequence-based changes such as single nucleotide polymorphisms (SNPs) or modifications such as m⁶A can alter RNA structure, which can in turn affect RNA function or protein binding. Non-coding RNAs can sequester proteins in specific cytoplasmic regions. RNA structures in UTRs can limit translation rates by impeding the initiation step. Despite the simple hairpin structures shown for clarity, cellular RNAs are known to adopt various complex structures. Adapted from Bevilacqua et al. 2016. Abbreviations: SNP, single nucleotide polymorphism; m⁶A, 6-methyladenosine; UTR, untranslated region.

Figure 2

The RNA processing effect induced by expanded repeats and representative structures of single expansions in select diseases. (a) In normal repeat RNA, splicing proteins and transcription factors are available for proper processing to mature mRNA isoforms. When repeats are expanded in disease, proteins needed for efficient splicing are sequestered, leading to excess of mis-spliced mRNA isoforms. Adapted from Todd and Paulson, 2010. (b) Secondary structures of repeat RNA and their associated diseases. Adapted from Blaszczyk et al, 2017. Abbreviations: N, nucleotide; HTT, Huntington gene; FMR1, Fragile X mental retardation 1; DMPK, DM1 protein kinase; ZNF9, Zinc finger protein 9.

Figure 3

Small molecule-based targeting of r(CUG)^exp repeats in DM1. (a) CTG expansion in the DMPK locus results in the transcription of r(CUG)^exp repeats that then sequester MBNL proteins, leading to some of the characteristic phenotypes in DM1. Abbreviations: DMPK, DM1 protein kinase; MBNL, Muscle-blind protein 1. (b) Representative small molecule inhibitors of steps in DM1 repeat pathogenesis.

Figure 4

Small molecule-based targeting of the miRNA biogenesis pathway. (a) Mechanism of action of miRNA-mediated gene silencing. (b) Example small molecule inhibitors of miRNA biogenesis. Abbreviations: pri-miRNA, primary miRNA; pre-miRNA, precursor miRNA; RISC, RNA-induced silencing complex.

Figure 5

Traditional and emerging screens to identify small molecule miRNA inhibitors. (a) A standard luciferase-based reporter system used to identify small molecule miRNA inhibitors. Increase in luciferase signal indicative of translation is assumed to be caused by small molecule binding to miRNA or one if its precursors, thereby reducing binding of the mature miRNA to its target sequence. Adapted from Mahato et al. (b) A click-chemistry-based assay to identify small molecule inhibitors of Dicer-mediated processing. Adapted from Garner et al. Abbreviations: RISC, RNA-induced silencing complex.

Figure 6

Structure, function, and small molecule binders of G-quadruplexes. (a) Nucleotide composition and base-pairing interactions in an RNA G-quadruplex and stacking of multiple G-quadruplexes. Adapted from Maiti et al. (b) Regulation of cap-dependent and independent translation by G-quadruplexes. Adapted from Balasubramanian et al. (c) Examples of small molecule stabilizers of G-quadruplexes in 5′-UTRs. Abbreviations: M+, monovalent metal cation; m⁷G, 7-methylguanylate; IRES, internal ribosomal entry site; NRAS, Neuroblastoma RAS viral oncogene homolog; KRAS, Kirsten rat sarcoma viral oncogene homolog; ss, splice site; Bcl-X, B-cell lymphoma-extra.

Figure 7

Small molecule-induced regulation of the SMN2 splicing mechanism. (a) The splicing mechanism of SMN1 (healthy protein) and SMN2 (disease protein) in which Exon 7 is included due to a SNP. (b) Small molecules shown to induce Exon 7 stabilization in SMN2 gene. Abbreviations: SNP, single nucleotide polymorphism; SMN, survival motor neuron; ESE, exonic splicing enhancer; hnRNP G, heterogeneous nuclear ribonucleoprotein G; snRNP, small nuclear ribonucleoprotein; ss, splice site.