RNA editing is a post-transcriptional process that modifies the genetic information on RNA molecules. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria. The PLS-type pentatricopeptide repeat (PPR) protein and the multiple organellar RNA editing factor (MORF, also known as RNA editing factor interacting protein (RIP)) are two types of key trans-acting factors involved in this process. However, how they cooperate with one another remains unclear. Here, we have characterized the interactions between a designer PLS-type PPR protein (PLS)PPR and MORF9, and found that RNA-binding activity of (PLS)PPR is drastically increased on MORF9 binding. We also determined the crystal structures of (PLS)PPR, MORF9 and the (PLS)PPR-MORF9 complex. MORF9 binding induces significant compressed conformational changes of (PLS)PPR, revealing the molecular mechanisms by which MORF9-bound (PLS)PPR has increased RNA-binding activity. Similarly, increased RNA-binding activity is observed for the natural PLS-type PPR protein, LPA66, in the presence of MORF9. These findings significantly expand our understanding of MORF function in plant organellar RNA editing.

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Background Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In most conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Methods Here, we developed a method of sequencing enzyme-accessible chromatin in circular DNA (CCDA-seq) based on the use of methylase to label open chromatin without fragmentation and exonuclease to enrich ecDNA sequencing depth, followed by long-read nanopore sequencing. Results Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding to ecDNA at a single-molecule resolution. Conclusions These results deepen the understanding of ecDNA regulatory mechanisms.

The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method “BIND&MODIFY” for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.

Background: Acute myocardial infarction (AMI) is one of the most common global causes of death.Although considerable progress has been made in AMI diagnosis, there remains an urgent need for novel diagnostic biomarkers for its prevention and treatment. Functional exosomal microRNAs (miRNAs) are recognized as potential biomarkers in many diseases. This study's objective was to identify specific plasma exosomal miRNAs with biomarker potential for early AMI detection.Methods: Exosomes from the plasma of 26 coronary artery disease (CAD) patients, 55 AMI patients, and 37 healthy controls were isolated and characterized by transmission electron microcopy (TEM), western blotting, and nanoparticle tracking analysis (NTA). The miRNAs were purified from exosomes, and unique molecular identifier (UMI) small RNA sequencing was performed. The random forest (RF) model was trained to predict potential biomarkers.Results: NTA demonstrated that nanoparticle concentration did not change after AMI, while nanoparticle size distribution significantly decreased. The CAD and AMI groups' miRNA expression profiles significantly differed from the healthy group's profile. The RF classifier could be used to distinguish the healthy group from the AMI group, but could not be used to distinguish the CAD group from the other groups, which caused a high classification error rate. Eighteen miRNAs were selected as biomarkers based on their RF classifier significance. The diagnostic accuracy of 18 miRNAs was evaluated using AUC values of 0.93, 0.87, and 0.75 to detect healthy controls, AMI, and CAD, respectively.Conclusions: Nanoparticle diameter and the 18 miRNAs may serve as simple and accessible fingerprints for early AMI diagnosis.