Sachithrani Umanda Madugalle

Extrachromosomal circular DNAs (ecDNAs) are double stranded, closed loop DNA molecules that act independently of the genome. Here we report the discovery of a brain-enriched ecDNA, ecCldn34d , that regulates the transcriptional state of plasticity-related genes and is temporally associated with the stability of fear memories. ecDNA therefore represents a novel class of mobile DNA elements that are involved in experience-dependent neuroadaptation.

The RNA modification N6-methyladenosine (m ⁶ A) is highly abundant in human brain and implicated in neurological disorders. Profiling m ⁶ A within RNA isoforms is a critical step toward understanding the complex mechanisms that underpin brain function and disease; however, we lack an isoform-level atlas of m ⁶ A sites in the brain. We applied Oxford Nanopore direct RNA sequencing (DRS) to three postmortem human brain regions—prefrontal cortex, caudate nucleus, and cerebellum—to simultaneously investigate the transcriptome and epitranscriptome at the isoform level. We identified 57,000 m ⁶ A sites within 15,000 isoforms, revealing both isoform- and brain region–specific patterning of m ⁶ A modifications. The prefrontal cortex exhibited a distinctive profile of specifically modified isoforms enriched in excitatory neurons and had the highest proportion of unannotated m ⁶ A sites. A population of isoforms were hypermodified and associated with excitatory neurons in all brain regions. Our results demonstrate the utility of isoform-level profiling of RNA modifications and provide insights into brain region specificity with implications for development and disease.

Primary liver cancer is the sixth most prevalent cancer globally and is often diagnosed late, when treatment options are limited. Secondary liver cancer, arising from metastasis of other cancers to the liver, is a common complication of advanced solid cancers and a significant cause of cancer-related morbidity and mortality. Existing treatment options for advanced primary and secondary liver tumours have limited efficacy and new treatment modalities have the potential to improve patient outcomes. mRNA therapeutics are readily delivered to the healthy liver after systemic administration, but their uptake and expression within liver tumours is unclear. Here we show that intravenous delivery of mRNA-LNPs efficiently transfects virtually all hepatocytes in healthy, fibrotic, and cirrhotic liver, and also many cells of spontaneous hepatocellular carcinomas in situ . Delivery of mRNA is also possible to xenograft models of both primary and secondary liver cancer, albeit with attenuated protein expression relative to the normal liver. These findings demonstrate the potential for systemically delivered mRNA-LNP therapies for liver disease and cancer.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation. One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

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Summary: The RNA modification N ⁶ -methyladenosine (m ⁶ A) is critically involved in the regulation of gene activity underlying experience-dependent plasticity, and is necessary for the functional interplay between RNA and RNA binding proteins (RBPs) in the nucleus. However, the complete repertoire of m ⁶ A-modified RNA interacting RBPs in the synaptic compartment, and whether they are involved in fear extinction, have yet to be revealed. Using RNA immunoprecipitation followed by mass spectrometry, we discovered 12 novel, synapsespecific, learning-induced m ⁶ A readers in the medial prefrontal cortex of male C57/B6 mice. m ⁶ A RNA-sequencing also revealed a unique population of learning-related m ⁶ A-modified RNAs at the synapse, which includes a variant of the long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 ( Malat1 ). m ⁶ A-modified Malat1 binds to a subset of novel m ⁶ A readers, including cytoplasmic FMR1 interacting protein 2 (CYFIP2) and dihydropyrimidase-related protein 2 (DPYSL2) and a cell-type-specific, state-dependent, and synapse-specific reduction in m ⁶ A-modified Malat1 disrupts the interaction between Malat1 and DPYSL2 and impairs fear extinction. The consolidation of fear-extinction memory therefore relies on an interaction between m ⁶ A-modified Malat1 and select RBPs in the synaptic compartment.