Boxuan Simen Zhao

Dynamic RNA-protein interactions influence RNA transport, storage, stability, and activity and are fundamental to a broad range of biological processes. During the oocyte-to-embryo transition (OET), fully differentiated gametes fuse and regain totipotency. Throughout the OET, the transcription remains silent. Spatiotemporal regulation of gene expression relies on the dynamic interactions between maternal transcripts and various protein complexes. Despite its fundamental importance, RNA-protein interaction dynamics at the global level during the OET remain poorly understood. Here, we leverage fractionation RNA-seq to profile RNA insolubility, a proxy for the interaction between RNAs and protein complexes, during zebrafish OET. We identify transcripts that transition between the soluble (relatively protein-free) and insoluble (heavily protein-associated) states and demonstrate that these phase transitions are driven by ribosome-dependent and ribosome-independent mechanisms. We demonstrate that the capacity of maternal RNAs to engage with protein complexes changes dynamically during the OET through remodeling of cis-regulatory features. We developed a machine-learning framework and uncovered multiple cis- and trans-regulatory features that may contribute to RNA insolubility. To validate these predictions, we examined poly(A) tail length and m6A dynamics. We found that poly(A) tail length is associated with both ribosome-dependent and ribosome-independent RNA phase transitions. In parallel, fractionation m6A-pulldown RNA-seq revealed highly dynamic changes in the m6A landscape during the OET. A correlation between m6A modifications and RNA insolubility is gradually established during the OET. Together, our findings uncover a multilayered regulatory mechanism governing RNA phase transitions during the OET, providing fundamental insights into post-transcriptional regulation of gene expression during development.

Physical proximity between RNA molecules, and between the proteins that bind them, underlies much of post-transcriptional regulation, yet the methods that convert these relationships into sequence do so at a cost. Established RNA-RNA interaction maps techniques depend on crosslinking, fragmentation, and/or proximity ligation, so each partner survives only as a short chimeric read in which full identity and sequence are not preserved. We describe PAIR-link (Priming Annealed Isothermal RNA linking), a ligation-free and fragmentation-free strategy that joins two separately transcribed RNAs into a single contiguous cDNA while retaining the sequence of both partners. Two engineered RNAs carry complementary tails at their 3' ends. These tails anneal and prime one another, and an isothermal Bst-family reverse transcriptase extends across the junction in both directions to copy both RNAs into one chimeric cDNA that can be amplified by PCR and read directly by Sanger sequencing. We first established the chemistry in vitro using synthesized oligonucleotides. Because the reaction requires a defined 3' end, we drove the barcode RNAs from RNA polymerase III promoters, whose transcripts terminate at a defined site, rather than from polyadenylated polymerase II transcripts. We then implemented PAIR-link in human cells as a reconstituted ribonucleoprotein in which each barcode RNA is tethered to one half of a split reporter protein (split-cpHaloTag or split-TurboID), so that reconstitution of the split protein brings the two RNAs together and facilitates linking. Bead-intact pull-down preserves the complex through reverse transcription and amplification. We recovered the expected sequence-verified chimeric product for reporters directed independently to the nucleus and to the cytoplasm, showing that the method operates across distinct subcellular environments. To our knowledge, PAIR-link is the first method to fuse two separately produced RNAs into a single chimeric cDNA molecule with full sequences of RNA preserved, without crosslinking, fragmentation, or ligation. We discuss the current scope of the approach, which is limited to RNAs with defined 3' ends, and outline routes toward future native transcripts and protein-interaction mapping.

Understanding neuronal connectivity at single-cell resolution remains a fundamental challenge in neuroscience, with current methods particularly limited in mapping long-distance circuits and preserving cell type information. Here we present Connectome-seq, a high-throughput method that combines engineered synaptic proteins, RNA barcoding and parallel single-nucleus and single-synaptosome sequencing to map neuronal connectivity at single-synapse resolution. This adeno-associated virus-based approach enables simultaneous capture of both synaptic connections and molecular identities of connected neurons. We validated this approach in the mouse pontocerebellar circuit, identifying both established and potentially uncharacterized synaptic connections. Through integrated analysis of connectivity and gene expression, we identified molecular markers enriched in connected neurons, suggesting potential molecular determinants of circuit-specific connectivity. By enabling systematic mapping of neuronal connectivity across brain regions with single-cell precision and gene expression information, Connectome-seq provides a scalable platform for comprehensive circuit analysis across different experimental conditions and biological states.

Understanding neuronal connectivity at single-cell resolution remains a fundamental challenge in neuroscience, with current methods particularly limited in mapping long-distance circuits and preserving cell type information. Here we present Connectome-seq, a high-throughput method that combines engineered synaptic proteins, RNA barcoding, and parallel single-nucleus and single-synaptosome sequencing to map neuronal connectivity at single-synapse resolution. This AAV-based approach enables simultaneous capture of both synaptic connections and molecular identities of connected neurons. We validated this approach in the mouse pontocerebellar circuit, identifying both established projections and potentially novel synaptic partnerships. Through integrated analysis of connectivity and gene expression, we identified molecular markers enriched in connected neurons, suggesting potential associated factors of circuit organization. By enabling systematic mapping of neuronal connectivity across brain regions with single-cell precision and gene expression information, Connectome-seq provides a scalable platform for comprehensive circuit analysis across different experimental conditions and biological states. This advance in connectivity mapping methodology opens new possibilities for understanding circuit organization in complex mammalian brains.

This review navigates the evolving landscape of N6‐methyladenosine (m6A) research approaches, emphasizing the importance of advanced technology in understanding RNA epigenetics. Beginning with the fundamentals of m6A and the need for high‐ throughput methods, the investigation progresses from low‐throughput approaches to high‐throughput technologies, encompassing antibody‐dependent and antibody‐free sequencing methods, as well as nanopore‐based direct mRNA sequencing and computation methods for m6A detection. Spatial techniques and imaging tools for m6A are also introduced in addition. The discussion of their special applications emphasizes the biological significance of absolute quantification, single‐nucleotide resolution, single‐molecule detection, and single‐cell profiling. The review concludes with a vision of ideal approaches that combine current technologies for comprehensive m6A sequencing, with the potential to further our understanding of gene regulation, cellular diversity, and their roles in health and disease.

Prof. Chuan He was awarded the Tetrahedron Prize this year, one of the world's most prestigious prizes in organic chemistry. This In Focus briefly delves into the remarkable work of Prof. Chuan He and explores how his recent accolades underscore his impact on the world of science. His seminal contributions have paved the way for new directions at the interface of organic chemistry and life sciences.

A monthly journal publishing high-quality, peer-reviewed research on all topics related to RNA and its metabolism in all organisms

N⁶-methyladenosine (m⁶A) is a prevalent post-transcriptional modification in mammalian mRNA. m⁶A is enriched in consensus sequences within long exons, near stop codons and at the 3′ untranslated regions (3′ UTRs). m⁶A is the first confirmed reversible mRNA modification with dedicated methyltransferases, demethylases and binding (effector) proteins. To date, four components of the m⁶A methyltransferase complex, two m⁶A demethylases and several m⁶A-binding proteins have been identified in mammals. m⁶A exerts its effects by directly recruiting effector proteins or by modulating RNA secondary structures, which modulate mRNA metabolism, including maturation, translation and decay. There is evidence to indicate that methylated transcripts can be sorted to synchronously fast track their metabolism. m⁶A has regulatory roles in many cellular processes, including circadian rhythm maintenance, stem cell differentiation and stress responses. m⁶A may facilitate cell-state transitions by regulating the metabolism of transcripts of key transcription factors. In addition to m⁶A, other modifications exist in mammalian mRNA, including N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C), pseudouridine and 2′-O-methylation (2′OMe). This collection of chemical modifications modulates nearly all aspects of RNA metabolism and related physiological processes, adding another layer to the already complex gene expression regulation pathways in eukaryotes, particularly in mammals.

5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.