RNA 5-methylcytidine (m 5 C) modification plays an essential role in regulating RNA metabolism and functions in cellular processes. Tools to achieve temporal and transcript-specific m 5 C editing for functional studies are still limited. Furthermore, methods enabling m 5 C editing triggered by specific cellular signals to study m 5 C functions are currently lacking. Here, we present a temporally and conditionally controlled m 5 C writing platform engineered through integrating abscisic acid (ABA)-mediated chemically induced proximity with split-dCas13b-NSUN2/NSUN6 technology. This system enables the writing of m 5 C by reconstituting the split dCas13b-based m 5 C editing complex at the guide RNA (gRNA)-targeted RNA transcript sites under the control of the inducer ABA. The deposition of m 5 C is inducible, reversible and selective. The deposited m 5 C is biologically active and influences the stability of endogenous mRNA transcripts. Moreover, by incorporating ABA prodrugs, the m 5 C writing can be triggered by signals associated with distinct physiological or disease conditions (e.g., tumor microenvironment and senescence). This conditional m 5 C editing strategy provides a new programmable tool for studying m 5 C biology in a context-dependent manner and expands the repertoire of RNA modification editing technologies.
Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet, in the widely used model organism, the budding yeast Saccharomyces cerevisiae, tRNA pseudouridine sites have not been comprehensively annotated. Here, we performed two high-throughput methods established for detecting tRNA pseudouridylation positions: Nanopore direct RNA sequencing (DRS) and 2-bromoacrylamide-assisted cyclization sequencing (BACS). Using DRS, we sequenced cytosolic tRNA from seven pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-two existing pseudouridine annotations and predicted an additional ten previously unannotated pseudouridine sites. Our analysis also revealed DRS signal changes at several non-uridine sites with the loss of a PUS, including apparent changes in modification abundances at position 37 upon deletion of Pus3. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3, underscoring the need for caution in interpreting DRS-based signal changes. Using BACS, we confirmed the ten DRS-predicted novel sites, while further detecting eleven additional previously unannotated pseudouridine sites. Combining existing modification annotations from the Modomics database with our DRS and BACS datasets, we created a map of all detected pseudouridines—totaling 126 sites, including 21 novel sites—and the enzymes responsible for their catalysis, across the forty-two nuclear-encoded S. cerevisiae tRNA isoacceptors. ### Competing Interest Statement R.A. is an inventor on a University of California patent licensed to Oxford Nanopore Technologies. M.J. is a consultant to Oxford Nanopore Technologies and has received reimbursement for travel, accommodation, and conference fees to speak at events. Other authors declare no competing interests. NIH Common Fund, R35 GM143125, R01 HG013876, T32GM007759, T32GM149387, HG010053 Donald E. and Delia B. Baxter Foundation, https://ror.org/03ht4bd53
PNLDC1 is a member of the DEDD deadenylases, known for its specificity in poly(A) tail removal during mRNA turnover. While initially characterized for its function in pre-piRNA trimming critical to spermatogenesis and transposon silencing, its broader deadenylase activity which is especially pronounced during early development and silenced during differentiation, remains poorly understood. In this study, we mapped the epigenetic regulation of Pnldc1 during mESC differentiation, uncovering its promoter methylation pattern associated with transcriptional repression. Pnldc1-knockout mESC models established via CRISPR/Cas9 were shown to retain viability and pluripotency. RNA-seq and subsequent polysome profiling revealed that Pnldc1 deficiency leads to the downregulation of gene networks involved in translation and a lower overall translational rate. Notably, PNLDC1 has been linked to epigenetic changes in various cancers and is associated with oncogenic signaling pathways. Using a homology model based on the related PARN deadenylase, we conducted virtual screening of compound libraries and selected 12 candidates for experimental testing. Experimental validation revealed that two compounds significantly impacted genes involved in stress response and signal transduction, while also suppressing aberrant proliferation. These findings highlight PNLDC1’s role in regulating mRNA stability during differentiation and proliferation and as a candidate for targeted therapeutic intervention.
G3BP1 is a regulator of stress granule assembly capable of driving condensation of RNA in vitro. Recently, we purified stress granule cores from yeast and mammalian cells, and found that in addition to RNA and proteins, they contain large amounts of circular double-stranded DNA (dsDNA). Since G3BP1 has been implicated in DNA-RNA heteroduplex resolution, we compared the ability of G3BP1 to drive condensation of cytoplasmic RNA, circular cytoplasmic dsDNA, and total nucleic acids (comprising both, circular dsDNA and RNA) extracted from purified stress granule cores of HEK293T cells. Imaging under near-physiologic conditions (1.7 mM MgCl2, no crowding agents, 0.1 ng/μL nucleic acids) revealed that G3BP1 most effectively induces condensation of the DNA-RNA mixture from stress granule cores. Comparison of the disruption of these condensates by treatment with RNase A, DNase, or RNase H further shows that they are especially susceptible to the latter two. The sensitivity to DNase and RNase H of G3BP1-induced condensates of the stress granule-derived nucleic acid mixtures underscores the importance of DNA in these cytoplasmic assemblies.
Errors in transcription generate transient RNA variants that are amplified through translation, with potential consequences for cellular phenotypes and RNA-based therapeutics. Accurate detection of these rare events relies on high-fidelity sequencing approaches such as Circle-Seq, but technical artifacts introduced during RNA preparation can distort error profiles and frequencies. Here, we systematically evaluate the impact of bacterial RNA sample preparation on Circle-Seq outputs. We show that RNA extraction and ribosomal RNA depletion introduce artifactual oxidative damage signatures, particularly C>U and G>A substitutions, that inflate apparent transcription error rates. Optimized mRNA preparation conditions substantially reduce these artifacts. In addition, we identify an RNase III fragmentation bias caused by its preference for structured RNAs, resulting in uneven transcriptome coverage. Chaotropic monovalent ions mitigate this bias and improve coverage uniformity. Finally, our optimized protocol captured, with improved accuracy, the frequency and spectrum of transcription errors in absence of the transcription fidelity factor GreA. Together, these results define the technical limitations of Circle-Seq and provide practical solutions to improve the accuracy and interpretability of transcription error measurements in bacterial systems.
