Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells.

Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.

Using sno-lncRNAs as potential markers for Prader-Willi syndrome diagnosis.

The genetic disorder Prader-Willi syndrome (PWS) is mainly caused by the loss of multiple paternally expressed genes in chromosome 15q11-q13 (the PWS region). Early diagnosis of PWS is essential for timely treatment, leading to effectively easing some clinical symptoms. Molecular approaches for PWS diagnosis at the DNA level are available, but the diagnosis of PWS at the RNA level has been limited. Here, we show that a cluster of paternally transcribed snoRNA-ended long noncoding RNAs (sno-lncRNAs, sno-lncRNA1-5) derived from the SNORD116 locus in the PWS region can serve as diagnostic markers. In particular, quantification analysis has revealed that 6,000 copies of sno-lncRNA3 are present in 1 µL whole blood samples from non-PWS individuals. sno-lncRNA3 is absent in all examined whole blood samples of 8 PWS individuals compared to 42 non-PWS individuals and dried blood samples of 35 PWS individuals compared to 24 non-PWS individuals. Further developing a new CRISPR-MhdCas13c system for RNA detection with a sensitivity of 10 molecules per µL has ensured sno-lncRNA3 detection in non-PWS, but not PWS individuals. Together, we suggest that the absence of sno-lncRNA3 represents a potential marker for PWS diagnosis that can be detected by both RT-qPCR and CRISPR-MhdCas13c systems with only microlitre amount of blood samples. Such an RNA-based sensitive and convenient approach may facilitate the early detection of PWS.

Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 in zebrafish.

Ikzf1 is a Krüppel-like zinc-finger transcription factor that plays indispensable roles in T and B cell development. Although the function of Ikzf1 has been studied extensively, the molecular mechanism underlying T lymphopoiesis remains incompletely defined during the embryonic stage. Here we report that the genetic ablation of ikzf1 in mutant zebrafish resulted in abrogated embryonic T lymphopoiesis. This was ascribed to impaired thymic migration, proliferation, and differentiation of hematopoietic stem/progenitor cells (HSPCs). Ccr9a and Irf4a, two indispensable factors in T lymphopoiesis, were the direct targets of Ikzf1 and were absent in the ikzf1 mutants. Genetic deletion of either ccr9a or irf4a in the corresponding mutant embryos led to obvious T cell development deficiency, which was mainly caused by disrupted thymic migration of HSPCs. Restoration of ccr9a in ikzf1 mutants obviously promoted HSPC thymus homing. However, the HSPCs then failed to differentiate into T cells. Additional replenishment of irf4a efficiently induced HSPC proliferation and T cell differentiation. Our findings further demonstrate that Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 and provide new insight into the genetic network of T lymphocyte development.

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

Efforts to advance RNA aptamers as a new therapeutic modality have been limited by their susceptibility to degradation and immunogenicity. In a previous study, we demonstrated synthesized short double-stranded region-containing circular RNAs (ds-cRNAs) with minimal immunogenicity targeted to dsRNA-activated protein kinase R (PKR). Here we test the therapeutic potential of ds-cRNAs in a mouse model of imiquimod-induced psoriasis. We find that genetic supplementation of ds-cRNAs leads to inhibition of PKR, resulting in alleviation of downstream interferon-α and dsRNA signals and attenuation of psoriasis phenotypes. Delivery of ds-cRNAs by lipid nanoparticles to the spleen attenuates PKR activity in examined splenocytes, resulting in reduced epidermal thickness. These findings suggest that ds-cRNAs represent a promising approach to mitigate excessive PKR activation for therapeutic purposes.

CRISPR-dCas13-tracing reveals transcriptional memory and limited mRNA export in developing zebrafish embryos.

BACKGROUND: Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export. RESULTS: We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression. CONCLUSIONS: This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.

Multi-color RNA imaging with CRISPR-Cas13b systems in living cells.

Visualizing RNA dynamics is important for understanding RNA function. Catalytically dead (d) CRISPR-Cas13 systems have been established to image and track RNAs in living cells, but efficient dCas13 for RNA imaging is still limited. Here, we analyzed metagenomic and bacterial genomic databases to comprehensively screen Cas13 homologies for their RNA labeling capabilities in living mammalian cells. Among eight previously unreported dCas13 proteins that can be used for RNA labeling, dHgm4Cas13b and dMisCas13b displayed comparable, if not higher, efficiencies to the best-known ones when targeting endogenous MUC4 and NEAT1_2 by single guide (g) RNAs. Further examination of the labeling robustness of different dCas13 systems using the GCN4 repeats revealed that a minimum of 12 GCN4 repeats was required for dHgm4Cas13b and dMisCas13b imaging at the single RNA molecule level, while >24 GCN4 repeats were required for reported dLwaCas13a, dRfxCas13d and dPguCas13b. Importantly, by silencing pre-crRNA processing activity of dMisCas13b (ddMisCas13b) and further incorporating RNA aptamers including PP7, MS2, Pepper or BoxB to individual gRNAs, a CRISPRpalette system was developed to successfully achieve multi-color RNA visualization in living cells.

lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription.

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperone-like mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production.

The caudal dorsal artery generates hematopoietic stem and progenitor cells via the endothelial-to-hematopoietic transition in zebrafish.

Zebrafish hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium of the ventral wall of the dorsal aorta (DA) through the endothelial-to-hematopoietic transition (EHT) from approximately 30 to 60 hours post fertilization (hpf). However, whether other artery sites can generate HSPCs de novo remains unclear. In this study, using live imaging and lineage tracing, we found that the caudal dorsal artery (CDA) in the caudal hematopoietic tissue directly gave rise to HSPCs through EHT. This process initiated from approximately 60 hpf and terminated at approximately 156 hpf. Compared with that in the DA, fewer EHT events were observed in the CDA. The EHT events in the DA and CDA were similarly regulated by Runx1 but differentially influenced by blood flow (i.e., the EHT frequency in CDA was affected to a lesser extent when circulation was compromised in the tnnt2a-/- mutant). Therefore, the whole artery, including both DA and CDA, was endowed with the ability to produce HSPCs during a much longer time period. Coincidently, the lineage tracing results indicated that adult hematopoietic cells originated from the embryonic endothelium, and those produced later preferentially colonized the adult thymus. Collectively, our study revealed that the CDA serves as an additional source of hematopoiesis, and it shows similar but not identical properties with the DA.