Association of Cannabis with Apneic Episodes in a Breastfed Infant: A Case Study.

Background: The use of cannabis and its perceived safety among pregnant and breastfeeding women has increased in the context of expanding legalization. Current guidelines recommend abstaining from the use of cannabis while pregnant or breastfeeding due to the potential for harm, although there is still much that is unknown in this field. Case Presentation: A 5-week-old infant presented with recurrent apneic episodes and a positive urine delta-9-tetrahydrocannabinol (THC) screening test. The infant's mother reported regular cannabis use for treatment of depression and anxiety while pregnant and breastfeeding. The infant was subsequently transitioned to formula feedings, and the infant's condition improved. Conclusion: Cannabis and its active metabolites can be transferred into breast milk and may have deleterious neurologic effects on infants. However, a causal relationship between cannabis exposure and short- or long-term neurologic sequelae has not yet been definitively established. Further studies are warranted to assess the safety of maternal cannabis use for breastfed infants.

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma.

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of an aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxoglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1α, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively affects the levels of N6-methyladenosine (m6A), while the levels of 5-formylcytosine (f5C) in mitochondrial tRNA are unaffected. This supports the hypothesis of a differential impact of fumarate accumulation on distinct RNA demethylases. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA.

Chemical modification of cytidine in noncoding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remain unknown due to a lack of sensitive site-specific sequencing technologies. Here, we report a protonation-dependent sequencing reaction for the detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human transfer RNA (tRNA). Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA and more broadly provide a molecular foundation for using optimized sequencing reactions to better understand the role of oxidized RNA cytidine residues in diseases.

Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA.

Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.

Arrow pushing in RNA modification sequencing.

Methods to accurately determine the location and abundance of RNA modifications are critical to understanding their functional role. In this review, we describe recent efforts in which chemical reactivity and next-generation sequencing have been integrated to detect modified nucleotides in RNA. For eleven exemplary modifications, we detail chemical, enzymatic, and metabolic labeling protocols that can be used to differentiate them from canonical nucleobases. By emphasizing the molecular rationale underlying these detection methods, our survey highlights new opportunities for chemistry to define the role of RNA modifications in disease.