Hironori Adachi

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation.

Nonsense mutations often result from single nucleotide substitutions that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of a gene. The impact of nonsense mutations is two-fold: (1) the PTC-containing mRNA is degraded by a surveillance pathway called nonsense-mediated mRNA decay (NMD) and (2) protein translation stops prematurely at the PTC codon, and thus no functional full-length protein is produced. As such, nonsense mutations result in a large number of human diseases. Nonsense suppression is a strategy that aims to correct the defects of hundreds of genetic disorders and reverse disease phenotypes and conditions. While most clinical trials have been performed with small molecules, there is an increasing need for sequence-specific repair approaches that are safer and adaptable to personalized medicine. Here, we discuss recent advances in both conventional strategies as well as new technologies. Several of these will soon be tested in clinical trials as nonsense therapies, even if they still have some limitations and challenges to overcome.

Ribonucleoproteins (RNPs), which are comprised of non-coding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP), which contributes to the biogenesis of small nuclear RNPs (snRNPs) that are central components of the spliceosome. Interestingly, three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here we provide evidence that these RNAs become part of novel RNPs we term regulatory RNPs (regRNPs). We postulate that regRNPs can impact rRNA modifications via interactions with the guide RNA component of small nucleolar RNPs (snoRNPs). Most modifications within rRNA (predominantly pseudouridylation and ribose 2’- O -methylation) are conducted by snoRNPs, and we hypothesize that the activity of at least some of these snoRNPs is under the control of regRNPs. Ribosome heterogeneity leading to specialized ribosomes is an exciting emerging concept. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides important and timely insight into how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus. Summary Statement: Processed scaRNAs give rise to a novel regulatory RNP which regulates the modification of ribosomal RNA. These findings provide insight into the mechanisms governing ribosome heterogeneity.