Unexplained stillbirth is hypothesised to occur due to premature placental ageing, with unexpected deterioration of placental function for gestational age. Circular RNAs (circRNAs) are enzyme resistant RNA molecules that accumulate in ageing tissues. Furthermore, circRNAs bind gDNA directly, forming circRNA:DNA complexes which can induce DNA breaks. This study investigated circRNA accumulation with gestational age in healthy and stillbirth placentae and determined whether circRNAs directly interact with placental DNA causing DNA damage. Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, 23, 26, 31, 34 weeks' gestation) were assessed for DNA damage using an alkaline Comet Assay. Expression levels of 6 candidate circRNAs (circ\_0009000, circ\_0024157, circ\_0061017, circ\_0036877, circ\_0054624 and circ\_0111277), and their linear transcripts, were quantified using qPCR. Physical interaction of candidate circRNAs with DNA was confirmed by DNA:RNA ImmunoPrecipitation (DRIP). The effect of circ\_0009000 knockdown in HEK293T cells was assessed following transfection with either a siRNA (designed to knockdown circ\_0009000) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. DNA damage was assessed by Comet Assay. Compared with earlier gestations (37, 38, 39 and 40 weeks'), placental DNA damage and expression of all 6 candidate circRNAs, but not their linear transcripts, were increased in 40 and 41+ weeks' gestation samples, and in stillbirth. DRIP-qPCR signal size was significantly larger in term placentae than in enzyme-treated controls, confirming that all candidate circRNA loci bind to placental DNA. Depletion of circ\_0009000 by specific siRNA in HEK293T cells, significantly reduced DNA damage compared to control. Stillbirth placentae show accelerated ageing with premature accumulation of candidate circRNAs (first evidence in humans) at levels consistent with older gestation tissue. Importantly, these circRNAs bind to DNA and circ\_0009000 causes DNA breaks in placenta. Therefore, circRNAs (circ\_0009000, circ\_0024157, circ\_0061017, circ\_0036877, circ\_0054624 and circ\_0111277) play a role in placental ageing and associate with stillbirth, likely via DNA damage. ### Competing Interest Statement Provisional patent #1297095, filed 2nd September 2024.

Circular RNAs (circRNAs) are a widespread, cell-, tissue-, and disease-specific class of largely non-coding RNA transcripts. These single-stranded, covalently-closed transcripts arise through non-canonical splicing of pre-mRNA, a process called back-splicing. Back-splicing results in circRNAs which are distinguishable from their cognate mRNA as they possess a unique sequence of nucleic acids called the backsplice junction (BSJ). CircRNAs have been shown to play key functional roles in various cellular contexts and achieve this through their interaction with other macromolecules, particularly other RNA molecules and proteins. To elucidate the molecular mechanisms underlying circRNA function, it is necessary to identify these interacting partners. Herein, we present an optimized strategy for the simultaneous purification of the circRNA interactome within eukaryotic cells, allowing the identification of both circRNA-RNA and circRNA-protein interactions.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington's disease (HD), results from expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of HTT pre-mRNA. The most abundant circRNA from HTT, circHTT(2-6), was found to be more highly expressed in the frontal cortex of HD patients, compared with healthy controls, and positively correlated with CAG repeat tract length. Furthermore, the mouse orthologue (mmu_circHTT(2-6)) was found to be enriched within the brain and specifically the striatum, a region enriched for medium spiny neurons that are preferentially lost in HD. Transgenic overexpression of circHTT(2-6) in two human cell lines-SH-SY5Y and HEK293-reduced cell proliferation and nuclear size without affecting cell cycle progression or cellular size, or altering the CAG repeat region length within HTT. CircHTT(2-6) overexpression did not alter total HTT protein levels, but reduced its nuclear localisation. As these phenotypic and genotypic changes resemble those observed in HD patients, our results suggest that circHTT(2-6) may play a functional role in the pathophysiology of this disease.

Circular RNAs (circRNAs) are covalently closed, single-stranded transcripts that are ubiquitously expressed in all eukaryotes and even prokaryotic archaea. Although once regarded as splicing artifacts, circRNAs are a novel class of regulatory molecules with diverse biological functions, including regulation of transcription, modulation of alternative splicing, and binding of miRNAs and proteins. The majority of studies of circRNAs have been performed in animals with a focus on the biogenesis, function, and mechanistic characterization of these molecules. In contrast, the study of circRNAs in plants is just emerging. Interestingly, recent circRNA profiling studies in model plant systems show distinct features of plant circRNAs compared with those from animals, including putative roles in stress response, differences in expression patterns, and novel biogenesis mechanisms. This provides a great opportunity to broaden our knowledge of circRNAs using plant model systems, such as Arabidopsis and rice, which are ideal for phenotypic characterization and genetic studies. In this review, we summarize current knowledge of plant circRNAs, discuss their identification and biogenesis, describe potential functions, and propose future perspectives for plant circRNA study.