Harnessing Nature’s Molecular Recognition Capabilities to Map and Study RNA Modifications

Abstract

Conspectus RNA editing or “epitranscriptomic modification” refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and although many are “silent” and do not change the Watson–Crick–Franklin code of the RNA, others result in recoding events. More than 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome. The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for the detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of the similar reactivity profiles of inosine and pseudouridine or other nucleobases. Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an “anti-inosine antibody” to develop new methods of detecting and enriching inosine from cellular RNA. Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.

Enzyme-mediated alkynylation enables transcriptome-wide identification of pseudouridine modifications

“…Natural enzymes often provide useful starting points to develop biotechnologies because of their exceptional substrate specificity and catalytic efficiency 25 . One major difference in physiochemical features between uridine and Ψ lies in an extra hydrogen donor at the N1 position of the Ψ nucleobase (Figure 1a).…”

“…To provide additional approaches for both in vitro and potential cell-based manipulation of Ψ, we turned to nature for inspirations on new labeling strategies for Ψ. Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications; exploiting these biomolecules offers a promising new direction for modulation of different RNA modifications 25 . After examining several enzymes for in vitro reactivity, we focused on a methyltransferase from Methanocaldococcus jannaschii 26 .…”

Tracking the Message: Applying Single Molecule Localization Microscopy to Cellular RNA Imaging

“…[9] This post-transcriptional enzymatic process alters the chemical structure of RNA and thus impacts translation efficiency and even protein sequence. [10] While most studies of RNA localization have focused on messenger RNA (mRNA), a growing body of evidence is showing that noncoding transcripts, such as long non-coding RNAs (lncRNAs) [11,12] and microRNAs (miRNAs) [13] perform crucial cellular functions that are strikingly dependent on their subcellular confinement. The regulatory elements responsible for this localization are still elusive, but these transcripts are manifestly enriched at specific spatiotemporal sites, suggesting the pivotal importance of asymmetric localization.…”

“…Further, the localization of a given transcript can impact the extent to which it undergoes RNA editing [9] . This post‐transcriptional enzymatic process alters the chemical structure of RNA and thus impacts translation efficiency and even protein sequence [10] . While most studies of RNA localization have focused on messenger RNA (mRNA), a growing body of evidence is showing that noncoding transcripts, such as long non‐coding RNAs (lncRNAs) [11,12] and microRNAs (miRNAs) [13] perform crucial cellular functions that are strikingly dependent on their subcellular confinement.…”