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Long non-coding RNAs (lncRNAs) associate with RNA-binding proteins (RBPs) to form lncRNA-protein complexes that act in a wide range of biological processes. Understanding the molecular mechanism of how a lncRNA-protein complex is assembled and regulated is key for their cellular functions. In this mini-review, we outline molecular methods used to identify lncRNA-protein interactions from large-scale to individual levels using bulk cells as well as those recently developed imaging and single-molecule approaches that are capable of visualizing RNA-protein assemblies in single cells and in real-time. Focusing on the latter group of approaches, we discuss their applications and limitations, which nevertheless have enabled quantification and comprehensive dissection of RNA-protein interactions possible.

Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR–RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d–BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR–RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels. This paper describes a CRISPR–Cas13 system to effectively target circRNAs and screen their functions in vitro and in vivo, which enables the study of relevant circRNA phenotypes in human cell proliferation and in mouse embryogenesis.

Although long noncoding RNAs (lncRNAs) are generally expressed at low levels, emerging evidence has revealed that many play important roles in gene regulation by a variety of mechanisms as they engage with proteins. Given that the abundance of proteins often greatly exceeds that of their interacting lncRNAs, quantification of the relative abundance, or even the exact stoichiometry in some cases, within lncRNA–protein complexes is helpful for understanding of the mechanism(s) of action of lncRNAs. We discuss methods used to examine lncRNA and protein expression at the single cell, subcellular, and suborganelle levels, the average and local lncRNA concentration in cells, as well as how lncRNAs can modulate the functions of their interacting proteins even at a low stoichiometric concentration.

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

SummaryCircular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing overlapped exons in circRNAs from those in linear cognate mRNAs1,2. Here we report that the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, effectively and specifically discriminates circRNAs from mRNAs, using guide (g)RNAs targeting sequences spanning the back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA,circFAM120A,promotes cell proliferationin vitroandin vivoby preventingFAM120AmRNA from binding the translation inhibitor IGF2BP2 for efficient translation. Application of RfxCas13d/BSJ-gRNA screening has also uncoveredcircMan1a2with regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.

Scutellaria baicalensis Georgi is an important medicinal plant used worldwide. Information about the genome of this species is important for scientists studying the metabolic pathways that synthesise the bioactive compounds in this plant. Here, we report a draft reference genome sequence for S. baicalensis obtained by a combination of Illumina and PacBio sequencing, which was assembled using 10 X Genomics and Hi-C technologies. We assembled 386.63 Mb of the 408.14 Mb genome, amounting to about 94.73% of the total genome size, and the sequences were anchored onto 9 pseudochromosomes with a super-N50 of 33.2 Mb. The reference genome sequence of S. baicalensis offers an important foundation for understanding the biosynthetic pathways for bioactive compounds in this medicinal plant and for its improvement through molecular breeding.