Biology subjects

Liang, Y.

Publications and source records attributed to Liang, Y..

At least 235 records · Page 13Linked to original sources

bioRxiv2022

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and reemergence strategy

O_LIOver 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high-quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high-quality transcripts and carried out RNA-seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza. Comparison of turion transcriptome to that of fronds, the actively growing leaf-like tissue, were carried out. C_LIO_LIBioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. C_LIO_LIWe identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as in pathways for starch and lipid utilization upon turion germination. Comparison of genome-wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. C_LIO_LISimilarities between turions and seeds provided evidence that key regulators for seed maturation and germination have been retooled for their function in turion biology. C_LI

plant biology↗

bioRxiv2022

Spateo: multidimensional spatiotemporal modeling of single-cell spatial transcriptomics

Cells do not live in a vacuum, but in a milieu defined by cell-cell communication that can be measured via emerging high-resolution spatial transcriptomics approaches. However, analytical tools that fully leverage such data for kinetic modeling remain lacking. Here we present Spateo (aristoteleo/spateo-release), a general framework for quantitative spatiotemporal modeling of single-cell resolution spatial transcriptomics. Spateo delivers novel methods for digitizing spatial layers/columns to identify spatially-polar genes, and develops a comprehensive framework of cell-cell interaction to reveal spatial effects of niche factors and cell type-specific ligand-receptor interactions. Furthermore, Spateo reconstructs 3D models of whole embryos, and performs 3D morphometric analyses. Lastly, Spateo introduces the concept of "morphometric vector field" of cell migrations, and integrates spatial differential geometry to unveil regulatory programs underlying various organogenesis patterns of Drosophila. Thus, Spateo enables the study of the ecology of organs at a molecular level in 3D space, beyond isolated single cells.

systems biology↗

bioRxiv2022

A high-quality reference genome for the fission yeast Schizosaccharomyces osmophilus

Fission yeasts are an ancient group of fungal species that diverged from each other from tens to hundreds of million years ago. Among them is the preeminent model organism Schizosaccharomyces pombe, which has significantly contributed to our understandings of molecular mechanisms underlying fundamental cellular processes. The availability of the genomes of S. pombe and three other fission yeast species S. japonicus, S. octosporus, and S. cryophilus has enabled cross-species comparisons that provide insights into the evolution of genes, pathways, and genomes. Here, we performed genome sequencing on the type strain of the recently identified fission yeast species S. osmophilus and obtained a complete mitochondrial genome and a nuclear genome assembly with gaps only at rRNA gene arrays. A total of 5098 protein-coding nuclear genes were annotated and orthologs for more than 95% of them were identified. Genome-based phylogenetic analysis showed that S. osmophilus is most closely related to S. octosporus and these two species diverged around 16 million years ago. To demonstrate the utility of this S. osmophilus reference genome, we conducted cross-species comparative analyses of centromeres, telomeres, transposons, the mating-type region, Cbp1 family proteins, and mitochondrial genomes. These analyses revealed conservation of repeat arrangements and sequence motifs in centromere cores, identified telomeric sequences composed of two types of repeats, delineated relationships among Tf1/sushi group retrotransposons, characterized the evolutionary origins and trajectories of Cbp1 family domesticated transposases, and discovered signs of interspecific transfer of two types of mitochondrial selfish elements.

genomics↗

bioRxiv2022

SapBase (Sapinaceae Genomic DataBase): a central portal for functional and comparative genomics of Sapindaceae species

Sapindaceae is a family of flowering plants, also known as the soapberry family, comprising 141 genera and about 1900 species (Pedro et al., 2010). Most of them are distributed in tropical and subtropical regions, including trees, shrubs, also woody or herbaceous vines. Some are dioecious, while others are monoecious. Many Sapindaceae species possess great economic value; some furnish delicious fruits, like lychee (Litchi chinensis), longan (Dimocarpus longan), rambutan (Nephelium lappaceum); and ackee (Blighia sapida) - the national fruit of Jamaica; some produce abundance secondary metabolites, like saponin from soapberry (Sapindus mukorossi), and seed oil from yellowhorn (Xanthoceras sorbifolium); some yield valuable timber including maple (Acer spp.) and buckeye (Aesculus glabra); and some are of great herbal medicinal value, like balloon-vine (Cardiospermum halicacabum). In the last decade, with the rocketing of next generation sequencing (NGS) and genomic technologies, the full genome sequences of several Sapindaceae plants have been resolved (Lin et al., 2017; Liang et al., 2019; Yang et al., 2019; Zhang et al., 2021; Hu et al., 2022; Xue et al., 2022). Among them, our recent publication of the lychee genome attracted broad attention (Edger, 2022; Hu et al., 2022; Lyu, 2022). Now the post-genome era arrives for Sapindaceae, however, there is no public genomic database available for any Sapindaceae species, let alone an integrative database for the whole Sapindaceae family. A unified data platform is in urgent need to collect, manage and share relevant data resources. Therefore, we integrated our home-brew NGS data with all publicly available data for seven Sapindaceae plants and constructed the Sapinaceae Genomic DataBase, named SapBase (www.sapindaceae.com), in order to provide genomic resources and an online powerful analytic platform for scientific research on Sapinaceae species and comparative studies with other plants.

genomics↗

bioRxiv2022

Inflammation-Responsive Micellar Nanoparticles from Degradable Polyphosphoramidates for Targeted Delivery to Myocardial Infarction

Nanoparticles that undergo a localized morphology change to target areas of inflammation have been previously developed but are limited by their lack of biodegradability. In this paper, we describe a low ring strain cyclic olefin monomer, 1,3-dimethyl-2-phenoxy-1,3,4,7-tetrahydro-1,3,2-diazaphosphepine 2-oxide (MePTDO), that rapidly polymerizes via ring-opening metathesis polymerization (ROMP) at room temperature to generate well-defined degradable polyphosphoramidates with high monomer conversion (>84%). Efficient MePTDO copolymerizations with norbornene-based monomers are demonstrated, including a norbornenyl monomer functionalized with a peptide substrate for inflammation-associated matrix metalloproteinases (MMPs). The resulting amphiphilic peptide brush copolymers self-assembled in aqueous solution to generate micellar nanoparticles (30 nm in diameter) which exhibit excellent cyto- and hemocompatibility and undergo MMP-induced assembly into micron scale aggregates. As MMPs are upregulated in the heart post-myocardial infarction (MI), the MMP-responsive micelles were applied to target and accumulate in the infarcted heart following intravenous administration in a rat model of MI. These particles displayed a distinct biodistribution and clearance pattern in comparison to non-degradable analogues. Specifically, accumulation at the site of MI, competed with elimination predominantly through the kidney rather than the liver. Together, these results suggest this as a promising new biodegradable platform for inflammation targeted delivery.

biochemistry↗

bioRxiv2022

Microglial process dynamics enhance neuronal activity by shielding GABAergic synaptic inputs

Microglia are resident immune cells of the central nervous system (CNS) and play key roles in brain homeostasis. During anesthesia, microglia increase their dynamic process surveillance and interact more closely with neurons. However, the functional significance of microglial process dynamics and neuronal interaction has remained unclear. Using in vivo two-photon imaging in awake mice, we discover that microglia enhance neuronal activity after the cessation of general anesthesia. Hyperactive neuron somata are directly contacted by microglial processes, which specifically co-localize with GABAergic boutons. Electron microscopy-based synaptic reconstruction after two-photon imaging reveals that microglial processes enter into the synaptic cleft to shield GABAergic inputs. Microglial ablation or loss of microglial {beta}2-adrenergic receptors prevent post-anesthesia neuronal hyperactivity. Together, our study demonstrates a previously unappreciated function of microglial process dynamics, which allow microglia to transiently boost neuronal activity by physically shielding inhibitory inputs.

neuroscience↗

bioRxiv2022

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

genomics↗

bioRxiv2022

HSPB1 facilitates chemoresistance through inhibiting ferroptotic cancer cell death and regulating NF-κB signaling pathway in breast cancer

Chemoresistance is one of the major causes of therapeutic failure and poor prognosis for breast cancer patients, especially for triple-negative breast cancer patients. However, the underlying mechanism remains elusive. Here, we identified novel functional roles of heat shock protein beta-1 (HSPB1), regulating the chemoresistance and ferroptotic cell death in breast cancer. Based on TCGA and GEO databases, HSPB1 expression was upregulated in breast cancer tissues and associated with poor prognosis of breast cancer patients, which was considered as an independent prognostic factor for breast cancer. Functional assays revealed that HSPB1 could promote cancer growth and metastasis in vitro and in vivo. Furthermore, HSPB1 facilitated doxorubicin resistance through protecting breast cancer cells from drug-induced ferroptosis. Mechanistically, HSPB1 could bind with Ik{beta}- and promote its ubiquitination-mediated degradation, leading to increased nuclear translocation and activation of NF-{kappa}B signaling. In addition, HSPB1 overexpression led to enhanced secretion of IL6, which further facilitated breast cancer progression. These findings revealed that HSPB1 upregulation might be a key driver to progression and chemoresistance through regulating ferroptosis in breast cancer, while targeting HSPB1 could be an effective strategy against breast cancer.

cancer biology↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for Label-Free Single-Cell Proteomics

The sensitivity of single-cell proteomics (SCP) has increased dramatically in recent years due to advances in experimental design, sample preparation, separations and mass spectrometry instrumentation. Further increasing the sensitivity of SCP methods and instrumentation will enable the study of proteins within single cells that are expressed at copy numbers too small to be measured by current methods. Here we combine efficient nanoPOTS sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in fast label-free analyses. WWA is based on data-dependent acquisition (DDA) but employs larger precursor isolation windows to intentionally co-isolate and co-fragment additional precursors along with the selected precursor. The resulting chimeric MS2 spectra are then resolved using the CHIMERYS search engine within Proteome Discoverer 3.0. Compared to standard DDA workflows, WWA employing isolation windows of 8-12 Th increases peptide and proteome coverage by ~28% and ~39%, respectively. For a 40-min LC gradient operated at ~15 nL/min, we identified an average of 2,150 proteins per single-cell-sized aliquots of protein digest directly from MS2 spectra, which increased to an average of 3,524 proteins including proteins identified with MS1-level feature matching. Reducing the active gradient to 20 min resulted in a modest 10% decrease in proteome coverage. We also compared the performance of WWA with DIA. DIA underperformed WWA in terms of proteome coverage, especially with faster separations. Average proteome coverage for single HeLa and K562 cells was respectively 1,758 and 1,642 based on MS2 identifications with 1% false discovery rate and 3042 and 2891 with MS1 feature matching. As such, WWA combined with efficient sample preparation and rapid separations extends the depths of the proteome that can be studied at the single-cell level.

cell biology↗

bioRxiv2022

Spatiotemporal orchestration of multicellular transcriptional programs and communications in spinal cord injury

While spinal cord injury (SCI) involves a complex cascade of cellular and pathological changes that last for months to years, the most dramatic and comprehensive molecular rewiring and multicellular re-organization occur in the first few days, which determine the overall progression and prognosis of SCI, yet remain poorly understood. Here, we resolved the spatiotemporal architecture of multicellular gene expression in a mouse model of acute SCI, and revealed the coordinated gene co-expression networks, the upstream regulatory programs, and in situ cell-cell interactions that underlay the anatomic disorganization as well as the immune and inflammatory responses conferring the secondary injury. The spatial transcriptomic analysis highlights that the genes and cell types in the white matter (WM) play a more active and predominant role in the early stage of SCI. In particular, we identified a distinct population of WM-originated, Igfbp2-expressing reactive astrocytes, which migrated to the grey matter and expressed multiple axon/synapse-supporting molecules that may foster neuron survival and spinal cord recovery in the acute phase. Together, our dataset and analyses not only showcase the spatially-defined molecular features endowing the cell (sub)types with new biological significance but also provide a molecular atlas for disentangling the spatiotemporal organization of the mammalian SCI and advancing the injury management.

neuroscience↗

bioRxiv2022

Heterosynaptic Plasticity of the Visuo-auditory Projection Requires Cholecystokinin released from Entorhinal Cortex Afferents

The entorhinal cortex is involved in establishing enduring visuo-auditory associative memory in the neocortex. Here we explored the mechanisms underlying this synaptic plasticity related to projections from the visual and entorhinal cortices to the auditory cortex, using optogenetics of dual pathways. High-frequency laser stimulation (HFLS) of the visuo-auditory projection did not induce long-term potentiation (LTP). However, after pairing with sound stimulus, the visuo-auditory inputs were potentiated following either infusion of cholecystokinin (CCK) or HFLS of the entorhino-auditory CCK-expressing projection. Combining retrograde tracing and RNAscope in situ hybridization, we show that CCK expression is higher in entorhinal cortex neurons projecting to the auditory cortex than in those originating from the visual cortex. In the presence of CCK, potentiation in the neocortex occurred when the pre-synaptic input arrived 200 ms before post-synaptic firing, even after just five trials of pairing. Behaviorally, inhibition of CCK signaling blocked the generation of associative memory. Our results indicate that neocortical visuo-auditory association is formed through heterosynaptic plasticity, which depends on release of CCK in the neocortex mostly from entorhinal afferents.

neuroscience↗

bioRxiv2022

Structure of mycobacterial respiratory Complex I

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found at low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel and suggests that menaquinone interacts more extensively than ubiquinone with a key catalytic histidine residue in the enzyme.

biochemistry↗

bioRxiv2022

A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta

Clinically licensed COVID-19 vaccines ameliorate viral infection by inducing vaccinee production of neutralizing antibodies that bind to the SARS-CoV-2 Spike protein to inhibit viral cellular entry (Walsh et al., 2020; Baden et al., 2021), however the clinical effectiveness of these vaccines is transitory as viral variants arise that escape antibody neutralization (Tregoning et al., 2021; Willett et al., 2022). Vaccines that solely rely upon a T cell response to combat viral infection could be transformational because they can be based on highly conserved short peptide epitopes that hold the potential for pan-variant immunity, but a mRNA-LNP T cell vaccine has not been shown to be sufficient for effective antiviral prophylaxis. Here we show that a mRNA-LNP vaccine based on highly conserved short peptide epitopes activates a CD8+ and CD4+ T cell response that prevents mortality in HLA-A*02:01 transgenic mice infected with the SARS-CoV-2 Beta variant of concern (B.1.351). In mice vaccinated with the T cell vaccine, 24% of the nucleated cells in lung were CD8+ T cells on day 7 post infection. This was 5.5 times more CD8+ T cell infiltration of the lungs in response to infection compared to the Pfizer-BioNTech Comirnaty(R) vaccine. Between days 2 and 7 post infection, the number of CD8+ T cells in the lung increased in mice vaccinated with the T cell vaccine and decreased in mice vaccinated with Comirnaty(R). The T cell vaccine did not produce neutralizing antibodies, and thus our results demonstrate that SARS-CoV-2 viral infection can be controlled by a T cell response alone. Our results suggest that further study is merited for pan-variant T cell vaccines, and that T cell vaccines may be relevant for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

immunology↗

bioRxiv2022

DHHC3 mediated Cadm4 palmitoylation regulates myelination in CNS

Cell adhesion molecule 4 (Cadm4) plays important roles on plasma membrane (PM) to regulate myelin formation and the downregulation of Cadm4 is a prominent feature in many demyelination diseases. However, how Cadm4 maintains its level on PM has been elusive. Here, we identify that Cadm4 is palmitoylated at cysteine-347 (C347) and palmitoylation regulates the stable localization of Cadm4 on PM, as blocking palmitoylation by mutating C347 into alanine (C347A) results in the dissociation of Cadm4 from PM and targeting for degradation. Intriguingly, blocking Cadm4 palmitoylation by introducing C347A (Cadm4-KI) causes myelin abnormalities in CNS, characterized by loss of myelination, myelin infoldings and hypermyelination. Moreover, it is uncovered that Cadm4 palmitoylation is catalyzed by DHHC3, reducing Cadm4 palmitoylation by the deletion of DHHC3 renders the redistribution of Cadm4 for degrading. Consistently, the genetic deletion of DHHC3 leads to downregulated Cadm4 palmitoylation and defects in CNS myelination, virtually phenocopies that of the Cadm4-KI mice. Our findings suggest a mechanism that the stable localization of Cadm4 on PM regulated by protein palmitoylation is vital for myelination in CNS.

neuroscience↗

bioRxiv2022

A Plastidial Glycolytic-Gluconeogenic Switch of Mitochondrial Origin Enables Diatom Adaptations to High Latitudes

Organic carbon fixed in chloroplasts through the Calvin Cycle can be diverted towards different metabolic fates, including cytoplasmic and mitochondrial respiration; gluconeogenesis; and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known in non-photosynthetic tissue. Here, we characterize a lower plastid glycolytic-gluconeogenesis pathway in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and PGAM (bis- phospho-glycerate mutase), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that it is expressed at elevated levels in high latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.

plant biology↗

bioRxiv2022

ID1 functions as an autonomous phosphate (Pi) regulator upstream of the miR399-ZmPHO2 signaling module in maize

The macronutrient phosphorus is essential for plant growth and development. Plants have evolved multiple strategies to increase the efficiency of Pi acquisition to protect themselves from Pi starvation. However, the crosstalk between Pi homeostasis and plant development remains to be explored. Here, we report that overexpressing microRNA399 in maize induced a premature aging syndrome after pollination. Knockout of ZmPHO2, one of the miR399 targets, resulted in a similar premature aging phenotype. Strikingly, we found that INDETERMINATE1 (ID1), a floral transition regulator, inhibits the transcription of ZmMIR399 genes by directly binding to their promoters, alleviating the repression of ZmPHO2 by miR399 and ultimately contributing to the maintenance of Pi homeostasis in maize. Unlike ZmMIR399 genes, whose expression is induced by Pi deficiency, ID1 expression is independent of the Pi status, indicating that ID1 is an autonomous regulator of Pi homeostasis. Furthermore, we found that ZmPHO2 was under selection during maize domestication, resulting in a more sensitive response to phosphate starvation in temperate maize than in tropical maize. Our study reveals a direct functional link between Pi-deprivation sensing by the miR399-ZmPHO2 regulatory module and plant developmental regulation by ID1.

plant biology↗

bioRxiv2022

Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking

We introduce herein a new strategy for intracellular RNA and DNA tracking that is robust, orthogonal and complementary to existing methods: Fluorogenic U-Rich Internal Loop (FLURIL) tagging with cell-permeable fluorophore-labeled bifacial Peptide Nucleic Acids (fbPNAs). Our approach uses an 8-nt (U4xU4) U-rich internal loop (URIL) in the RNA of interest (ROI) as a compact labeling site for fluorogenic triplex hybridization with a bPNA probe (~1 kD). FLURIL tagging thus replaces a 4 bp duplex stem with a labeled 4-base-triple hybrid stem of similar structure and mass. In contrast to existing strategies for RNA tracking, FLURIL tagging can be applied to internal, genetically encoded URIL RNA sites with minimal structural perturbation, co-expression of protein-fusion labels or significant increase in molecular weight and steric bulk. We demonstrate effective FLURIL tagging of intracellular (HEK-293) RNAs, ribonucleoprotein (RNP) complexes and live cell (U2-OS) tracking of genomic loci. FLURIL tracking was internally validated by direct comparison with the most widely used live-cell RNA labeling method, MS2-labeling with MCP-HaloTag and Janelia Fluor dyes. In addition, FLURIL-tagging correctly reported on the endogenous RNP in HEK293 cells formed from TAR DNA binding protein 43 (TDP-43-tdTomato) and UG repeat RNA. The FLURIL strategy was also successfully applied to guide RNA (gRNA) in CRISPR-dCas complexes to enable live cell tracking of a low-copy number genomic locus (IDR3), internally benchmarked against MS2/HaloTag labeling of CRISPR-Sirius gRNA targeted to a proximal locus (IDR2). Notably, FLURIL-tagged IDR2 exhibited similar brightness as loci targeted by CRISPR-Sirius gRNA complexes, which bear 8-MS2 hairpins for protein labeling. Together, these experiments show that FLURIL tagging can simply and reliably track intracellular RNA, RNPs, and DNA, with a streamlined molecular footprint relative to other methods. Importantly, these data also indicate that FLURIL tagging is fully compatible with existing labeling methods without crosstalk and may be used to broaden the scope of intracellular RNA and DNA tracking. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/501035v1_figS1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e6cc24org.highwire.dtl.DTLVardef@1646707org.highwire.dtl.DTLVardef@a80268org.highwire.dtl.DTLVardef@363193_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO FLURIL-tagging of RNAs with bPNA probes. (a) Triplex hybridization of a U-rich internal loop (URIL) with bPNA (blue) via base triple formation between the melamine base (M) and two uracil bases (inset). (b) General schematic of labeling strategy described herein. An RNA of interest is engineered to contain an URIL and expressed within the cell, with a fluorogenic bPNA probe introduced via cell culture media. Successful URIL targeting is reported by an increase in emission (green) and confirmed by a previously established RNA binding protein with a fluorescent protein (red) fusion. C_FIG

cell biology↗