Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates. 

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient

Circular RNA in cancer

In 2012, it was discovered that precise gene editing could be induced in target DNA using the reprogrammable characteristics of the CRISPR system. Since then, several studies have investigated the potential of the CRISPR system to edit various biological organisms. For the typical CRISPR system obtained from bacteria and archaea, many application studies have been conducted and have spread to various fields. To date, orthologs with various characteristics other than CRISPR-Cas9 have been discovered and are being intensively studied in the field of gene editing. CRISPR-Cas12 and its varied orthologs are representative examples of genome editing tools and have superior properties in terms of in vivo target gene editing compared with Cas9. Recently, TnpB and Fanzor of the OMEGA (obligate mobile element guided activity) system were identified to be the ancestor of CRISPR-Cas12 on the basis of phylogenetic analysis. Notably, the compact sizes of Cas12 and OMEGA endonucleases allow adeno-associated virus (AAV) delivery; hence, they are set to challenge Cas9 for in vivo gene therapy. This review is focused on these RNA-guided reprogrammable endonucleases: their structure, biochemistry, off-target effects, and applications in therapeutic gene editing.

Recent application of CRISPR-Cas12 and OMEGA system for genome editing.

Nuclear bodies (NBs) are biomolecular condensates that participate in various cellular processes and respond to cellular stimuli in the nucleus. The assembly and function of these protein- and RNA-rich bodies, such as nucleoli, nuclear speckles, and promyelocytic leukemia (PML) NBs, contribute to the spatial organization of the nucleus, regulating chromatin activities locally and globally. Recent technological advancements, including spatial multiomics approaches, have revealed novel roles of nucleoli in modulating ribosomal DNA (rDNA) and adjacent non-rDNA chromatin activity, nuclear speckles in scaffolding active genome architecture, and PML NBs in maintaining genome stability during stress conditions. In this review, we summarize emerging functions of these important NBs in the spatial organization of the genome, aided by recently developed spatial multiomics approaches toward this direction. 

Emerging roles of nuclear bodies in genome spatial organization.

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr. 

Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle.

Nucleolar URB1 ensures 3′ ETS rRNA removal to prevent exosome surveillance

Therapeutic application of circular RNA aptamers in a mouse model of psoriasis.

<h2>Abstract</h2> Numerous circular RNAs (circRNAs) produced from back-splicing of exon(s) have been recently revealed on a genome-wide scale across species. Although generally expressed at a low level, some relatively abundant circRNAs can play regulatory roles in various biological processes, prompting continuous profiling of circRNA in broader conditions. Over the past decade, distinct strategies have been applied in both transcriptome enrichment and bioinformatic tools for detecting and quantifying circRNAs. Understanding the scope and limitations of these strategies is crucial for the subsequent annotation and characterization of circRNAs, especially those with functional potential. Here, we provide an overview of different transcriptome enrichment, deep sequencing and computational approaches for genome-wide circRNA identification, and discuss strategies for accurate quantification and characterization of circRNA. 

Approaches and challenges in genome-wide circular RNA identification and quantification.

We develop a stepwise computational framework, called DEMINING, to directly detect expressed DNA and RNA mutations in RNA deep sequencing data. DEMINING incorporates a deep learning model named DeepDDR, which facilitates the separation of expressed DNA mutations from RNA mutations after RNA-seq read mapping and pileup. When applied in RNA-seq of acute myeloid leukemia patients, DEMINING uncovered previously-underappreciated DNA and RNA mutations, some associated with the upregulated expression of host genes or the production of neoantigens. Finally, we demonstrate that DEMINING could precisely classify DNA and RNA mutations in RNA-seq data from non-primate species through the utilization of transfer learning.

A deep learning model embedded framework to distinguish DNA and RNA mutations directly from RNA-seq

Back-splicing of eukaryotic exon(s) leads to the production of covalently closed circular RNAs (circRNAs). Generally, most circRNAs contain overlapping sequences to their cognate linear RNAs from the same gene loci, leading to difficulties in distinguishing them from each other. A recent study has shown that some circRNAs can be specifically depleted by using base editing systems to target their predominantly back-splice sites for circularization, suggesting an efficient approach for circRNA knockout (KO). Here, we describe the detailed protocol for applying base editors to disrupt back-splice sites of predominantly circularized exons for circRNA KO at the genomic DNA level. 

Base-Editor-Mediated circRNA Knockout by Targeting Predominantly Back-Splice Sites.

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