The classical view of gene regulation draws from prokaryotic models, where responses to environmental changes involve operons regulated by sequence-specific protein interactions with DNA, although it is now known that operons are also modulated by small RNAs. In eukaryotes, pathways based on microRNAs (miR) regulate the readout of genomic information from transcripts, while alternative nucleic acid structures encoded by flipons influence the readout of genetic programs from DNA. Here, we provide evidence that miR- and flipon-based mechanisms are deeply connected. We analyze the connection between flipon conformation and the 211 highly conserved human miR that are shared with other placental and other bilateral species. The direct interaction between conserved miR (c-miR) and flipons is supported by sequence alignments and the engagement of argonaute proteins by experimentally validated flipons as well as their enrichment in promoters of coding transcripts important in multicellular development, cell surface glycosylation and glutamatergic synapse specification with significant enrichments at false discovery rates as low as 10−116. We also identify a second subset of c-miR that targets flipons essential for retrotransposon replication, exploiting that vulnerability to limit their spread. We propose that miR can act in a combinatorial manner to regulate the readout of genetic information by specifying when and where flipons form non-B DNA (NoB) conformations, providing the interactions of the conserved hsa-miR-324-3p with RELA and the conserved hsa-miR-744 with ARHGAP5 genes as examples.

Identifying roles for Z-DNA remains challenging given their dynamic nature. Here, we perform genome-wide interrogation with the DNABERT transformer algorithm trained on experimentally identified Z-DNA forming sequences (Z-flipons). The algorithm yields large performance enhancements (F1 = 0.83) over existing approaches and implements computational mutagenesis to assess the effects of base substitution on Z-DNA formation. We show Z-flipons are enriched in promoters and telomeres, overlapping quantitative trait loci for RNA expression, RNA editing, splicing, and disease-associated variants. We cross-validate across a number of orthogonal databases and define BZ junction motifs. Surprisingly, many effects we delineate are likely mediated through Z-RNA formation. A shared Z-RNA motif is identified in SCARF2, SMAD1, and CACNA1 transcripts, whereas other motifs are present in noncoding RNAs. We provide evidence for a Z-RNA fold that promotes adaptive immunity through alternative splicing of KRAB domain zinc finger proteins. An analysis of OMIM and presumptive gnomAD loss-of-function datasets reveals an overlap of Z-flipons with disease-causing variants in 8.6% and 2.9% of Mendelian disease genes, respectively, greatly extending the range of phenotypes mapped to Z-flipons.

Identifying roles for Z-flipons remains challenging given their dynamic nature. Here we perform genome-wide interrogation with the DNABERT transformer algorithm trained on experimentally identified Z-DNA sequences. We show Z-flipons are enriched in promoters and telomeres and overlap quantitative trait loci for RNA expression, RNA editing, splicing and disease associated variants. Surprisingly, many effects are mediated through Z-RNA formation. We describe Z-RNA motifs present in SCARF2, SMAD1 and CACNA1 transcripts and others in non-coding RNAs. We also provide evidence for another Z-RNA motif that likely enables an adaptive anti-viral intracellular defense through alternative splicing of KRAB domain zinc finger proteins. An analysis of OMIM and gnomAD predicted loss-of-function datasets reveals an overlap of predicted and experimentally validated Z-flipons with disease causing variants in 8.6% and 2.9% of mendelian disease genes respectively, with frameshift variants present in 22% of cases. The work greatly extends the number of phenotypes mapped to Z-flipon variants.

The classical view of gene regulation is based on prokaryotic models and the operon concept with protein-based transcription factors controlling the expression of metabolic pathways essential for bacterial adaptations in response to environmental changes. A new view for establishing cell identity is emerging in eukaryotes where RNA-based pathways provide the framework for the readout of genomic information. Another perspective poses that alternative DNA structures encoded by flipons enable switching of cellular responses from one state to another. Here we provide evidence that these RNA and DNA mechanisms are deeply connected. We present data supporting a model where flipons open up binding sites for microRNAs (miRNAs), leading to the establishment of bivalent promoters early in development whose location structures lineage-specific events. These outcomes are potentially influenced by ovarian and spermatozoan miRNAs, transmissions with evident evolutionary ramifications. The data supports a new perspective on genetic regulation, one in which the genome provides a canvas framed by flipons, sketched with miRNAs and embellished by proteins.