5-bromouridine Research Articles

Cell Surface RNAs Control Neutrophil Function

Recently, RNAs localizing to the outer cell surface have been reported in mammalian cells, with such RNAs containing glycan modifications (referred to as GlycoRNAs). However, the function of cell surface RNAs are poorly known. In this study, we investigated whether cell surface RNAs exist in neutrophils, what their functions are, and the mechanism by which they function. We first determined that neutrophils express cell surface GlycoRNAs. We utilized two strategies to assess cell surface GlycoRNAs. First, we utilized a sialic acid homologue to metabolically label glycans inside cells. Labeled glycans can be readily detected in purified total RNAs from primary murine neutrophils. The GlycoRNAs signals were depleted upon RNase digestion but not digestion by proteinase or DNase, supporting the existence of GlycoRNAs in neutrophils. Importantly, treating live neutrophils with RNase extracellularly removed over 90% of GlycoRNA signals, supporting that the majority of GlycoRNAs were located on the surface of neutrophils. Second, we directly visualized cell surface RNAs by labeling cellular RNAs with the nucleoside homologue 5'-bromouridine (BrU) and detecting live cells with an anti-BrU antibody applied extracellularly. These data support the existence of cell surface GlycoRNAs on neutrophils. We next revealed that cell surface GlycoRNAs play important functions in neutrophils to mediate transendothelial migration both in vivo and in vitro. We utilized an acute peritonitis model in which primarily neutrophils with cell surface GlycoRNAs removed by extracellular RNase were injected into circulation in mice that were treated with thioglycolate, and the migration of these neutrophils into the peritoneal cavity was quantified and compared to mock treated cells in vivo. We observed a 9-fold decrease of migration by neutrophils treated with extracellular RNase. To determine the in vitro function of cell surface RNAs, we tested the ability of neutrophils to migrate toward a chemoattractant in a trans-well assay. While neutrophils treated with extracellular RNase were viable and migrated similarly as control neutrophils, we observed a substantial defect in migration by extracellular-RNase-treated neutrophils when an endothelial layer was present on the tanswell membrane. A similar defect was observed when assaying neutrophil attachment to endothelial cells in vitro. This defect can be replicated using control neutrophils but by pre-blocking endothelial cells with purified neutrophil GlycoRNAs or the glycan fraction of GlycoRNAs. The defect in neutrophil endothelial interaction in vivo was observed by intravital confocal microscopy. These data support the function of neutrophil cell surface GlycoRNAs in helping transendothelial migration. Lastly, we found that neutrophil GlycoRNAs are bona fide ligands for P-selectin on endothelial surface. Removal of neutrophil surface RNAs with extracellular RNase treatment did not significantly change cell surface integrin levels or reactivity, but reduced recombinant P-selectin binding. Furthermore, recombinant P-selectin, but not recombinant E-selectin, can detect glycoRNAs in purified neutrophil total RNAs. Additionally, blocking endothelial cells with an antibody against P-selectin led to a significant reduction in GlycoRNA binding. These data support that GlycoRNA-P-selectin interaction, at least in part, mediate neutrophil transendothelial migration. Our data demonstrate a critical role of cell surface RNAs in neutrophils, and reveal a new dimension that regulate the function of hematopoietic cells.

5'-Bromouridine IP Chase (BRIC)-Seq to Determine RNA Half-Lives.

Analysis of RNA stability at genome-wide level is an advanced method in RNA biology that examines the half-life of each transcript. In particular, a pulse-labeling method using uridine analogs enables the determination of half-life of each transcript under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromouridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq.

Abstract P6-08-05: Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq

Abstract Estrogen promotes estrogen receptor (ER)-positive breast cancer cell growth by modulating transcription through ligand activated ER. The spectrum of early ER target genes has been identified through gene expression microarray and more recently with RNA-seq analyses. These methods, although comprehensive, fall short in identifying many ER-regulated transcripts which are either not present in microarray; too abundant to determine estrogen regulated changes among steady-state pools of RNAs; or have very short half-lives and low abundance such as some non-coding RNAs, including RNAs generated by enhancers bound by ER (eRNAs). Global run-on sequencing (GRO-seq) has been used to identify ER target genes and eRNAs in breast cancer cells. It is technically cumbersome and is performed under non-physiological condition. We have applied BrU-seq technique to identify global transcripts regulated by estrogen in MCF-7 breast cancer cells. The newly synthesized RNAs in MCF-7 cells treated with or without estrogen for various time points, were labeled with 5'-bromo-uridine (BrU). The labeled RNAs were purified by immunoprecipitation using an anti-BrdU antibody and then subjected to RNA sequencing analysis, which provides greater sensitivity than regular RNA-seq analysis from steady-state total RNA samples. Robust estrogen-regulated transcripts can be detected within 30 min of treatment. Significantly more transcripts (both coding and non-coding RNAs) were identified as estrogen targets using BrU-seq analysis than RNA-seq analysis from total RNA pools. Many of these estrogen-regulated transcripts are located close to ER binding sites identified from ER ChIP-seq analysis, indicating a functional role for these ER binding sites in regulating RNA transcription. For estrogen targets identified through both analyses, the magnitude of expression changes (both up- and down-regulation) is usually greater from BrU-seq than from the regular RNA-seq analysis. The dynamics of RNA synthesis and degradation for some unstable RNAs (such as eRNAs) can be interrogated through our data set. Also, RNA pol II elongation rate can be deduced from some long ER target genes (>100 kb) such as GREB1. In addition to identifying estrogen targets, BrU-seq should be a very useful technique in assessing transcriptional changes triggered by various signal transduction pathways. Citation Format: Sun J, Capobianco E, Tsinoremas N, Lippman M. Genome-wide identification of transcripts regulated by estrogen in MCF-7 cells using BrU-seq [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-05.

Genome-wide analysis of long noncoding RNA turnover.

Genome-wide analysis for determining RNA turnover is an advanced method in RNA biology that examines the specific half-life of nuclear noncoding RNA (ncRNA). In particular, a pulse-labeling method using uridine analogs enables the determination of RNA stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing. The method is called BrU immunoprecipitation chase assay (BRIC) or BRIC through deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq.

BRIC-seq: A genome-wide approach for determining RNA stability in mammalian cells

We recently developed a novel transcriptome analysis method, termed 5′-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis (BRIC-seq). BRIC-seq enables the determination of genome-wide RNA stability by chasing chronological decreases of BrU-labeled RNAs under physiologically undisturbed conditions. The RNA half-life of each transcript is calculated from the decreasing number of BrU-labeled RNA sequence tags measured by deep sequencing of BrU-labeled RNAs. Here, we describe a detailed protocol and provide tips for BRIC-seq, followed by computational analysis.

SOX2 Modulates Reprogramming of Gene Expression in Two-Cell Mouse Embryos1

Sox2 is a key gene that controls transcriptional networks required for pluripotency. The role of Sox2 in the developmental transition of a highly differentiated oocyte to totipotent blastomeres of the early preimplantation embryo, however, is not known. We report that Sox2, which is localized in the nucleus, is first zygotically expressed during the 2-cell stage and that its expression dramatically increases between the morula and blastocyst stages. Injecting a cRNA encoding Sox2 into 1-cell embryos resulted in overexpression of SOX2 by approximately 70% and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, Myc (also known as c-Myc), or Klf4 has little effect on the ability of 2-cell embryos to cleave to the 4-cell stage. Global transcription assessed by bromo uridine triphosphate incorporation is reduced by approximately 15%, and transcript profiling revealed that approximately 15% of zygotically expressed genes are dramatically repressed in 2-cell embryos overexpressing SOX2. Furthermore, overexpressing a dominant-negative SOX2 perturbs reprogramming of gene expression in 2-cell embryos, though to a much lesser extent than that observed following overexpression of SOX2, and leads to developmental failure after the 2-cell stage but before the 8-cell stage. Results of these experiments implicate Sox2 as a critical transcriptional regulator in the oocyte-to-embryo transition that entails formation of totipotent blastomeres and indicate that the amount of Sox2 is critical for successful execution of this transition.

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Visualization of transcription sites at the electron microscope.

In order to localize at EM level the sites of transcription of both pre-mRNA and pre-rRNA, we have detected the DNA/RNA hybrid molecules and m3Gcapped structures by means of specific antibodies after short bromo-uridine (BrU) incorporation. In addition, the sections have been stained by a selective RNA stain, terbium citrate. Our data indicate that perichromatin fibrils incorporate BrU and are labeled by the anti-hybrid probe; this supports the idea that they are the pre-mRNA transcription sites. On the contrary, interchromatin granules do not incorporate BrU after short pulses and are not labeled by the anti-hybrid probe. Concerning the nucleolus, anti-hybrid and anti-BrdU antibodies colocalize only on the dense fibrillar component, suggesting that this is the site of rRNA transcription. Interestingly, the dense fibrillar component and the granular component, after specific RNA staining, show remarkable structural similarities, both containing fibrogranular RNA structures.

Non‐radioisotope method for diagnosing photosensitive genodermatoses and a new marker for xeroderma pigmentosum variant

Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by photo-induced deterioration of the skin, which often leads to the early development of skin cancers. To diagnose patients with XP and the related disorder Cockayne syndrome (CS), our laboratory has established a simple autoradiographic method that examines three cellular markers of DNA repair: unscheduled DNA synthesis (UDS), recovery of RNA synthesis (RRS) and recovery of replicative DNA synthesis (RDS). However, it is very laborious to measure the three markers using tritiated thymidine or uridine; therefore, we developed a non-isotope method for diagnosing XP and CS. Fibroblasts from the patient were labeled with bromodeoxyuridine (BrdU) instead of tritiated thymidine to measure UDS and RDS, or were labeled with bromouridine (BrU) instead of tritiated uridine to measure RRS. Incorporated BrdU or BrU could be detected using the immunofluorescence method. Moreover, we discovered a new useful marker for XP variant based on checkpoint activity. The non-radioisotope method and the new marker described here comprise an easy way to diagnose XP and CS.

Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms.

Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.

The architecture of interphase chromosomes and nucleolar transcription sites in plants

Fluorescence in situ hybridization (FISH) coupled with confocal microscopy has been used to reveal the interphase chromosome organization in plants. In wheat and several other related species, we have shown that the interphase chromosomes are in a very well-defined organization, with centromeres and telomeres located at opposite sides of the nuclear envelope—a classic Rabl configuration. In transgenic wheat lines, FISH analysis of metaphase chromosomes has shown that multiple transgene copies can be integrated along a single chromosome, with large regions of intervening genomic sequence. These multiple copies are often colocalized in interphase, suggesting either an ectopic association or a highly reproducible interphase chromatin configuration. Bromo-uridine (BrU) incorporation has been used to label transcription sites in the nucleolus. Using pea root tissue, we have combined BrU incorporation with preembedding 1-nm gold detection to image the nucleolar transcription sites by electron microscopy. This has revealed many distinct elongated clusters of silver–gold particles. These clusters are 200–300 nm in length and are thicker at one end than the other. We suggest that each cluster corresponds to a single transcribed gene. Serial sectioning of several entire nucleoli has enabled the reconstruction of all the nucleolar transcription sites, and we have estimated that there are 200–300 transcribed genes per nucleolus.

Nascent Flavivirus RNA Colocalizedin Situwith Double-Stranded RNA in Stable Replication Complexes

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitoredin situby immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G.,Virology140, 68–79, 1985). The model assumes that there is on average only one nascent strand per template, which remains bound until displaced during the next cycle of RNA synthesis. The replicase complex located by BrU incorporation in the identified foci was stable, remaining active in incorporating BrU or [32P]orthophosphate in viral RNA after complete inhibition of protein synthesis in cycloheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immunogold labeling of induced vesicle packets is functioning as the true replicative intermediate (Westawayet al., J. Virol.71, 6650–6661, 1997; Mackenzieet al., Virology245, 203–215, 1998). Implications are that the replicase complex is able to recycle in the same membrane site in the absence of continuing protein synthesis and that possibly apart from uncleaved NS3–NS4A, it has no requirement for a polyprotein precursor late in infection.

Mechanistic Studies Relevant to Bromouridine‐Enhanced Nucleoprotein Photocrosslinking: Possible Involvement of an Excited Tyrosine Residue of the Protein

AbstractThe results of mechanistic studies on formation of uridine (U) and N‐acetyl‐in‐(5‐uridinyl)tyrosine N‐ethylam‐ide (2) from irradiation of aqueous, pH 7 solutions of bromouridine (BrU) and N‐acetyltyrosine JV‐ethylamide (1) are reported. Solutions were irradiated with monochromatic laser emission at 266, 308 and 325 nm. Quantum yield measurements as a function of excitation wavelength suggest that both products result from excitation of the tyrosine derivative followed by electron transfer to BrU, possibly with intermediacy of the hydrated electron. The BrU radical anion ejects bromide to form the uri‐dinyl radical, which then abstracts a hydrogen atom from 1 or adds to the aromatic ring of 1. Formation of adduct 2 is a model for photocrosslinking of nucleic acids bearing the bromouracil chromophore to adjacent tyrosine residues of proteins in nucleoprotein complexes. The value of 325 nm excitation in photocrosslinking, where the tyrosine chromophore is more competitive for photons, was demonstrated with an RNA bound to the MS2 bacteriophage coat protein; more than a 60% increase in the yield of photocrosslinking relative to that obtained with 308 nm excitation was achieved.

In vitro splicing of pre-mRNA containing bromouridine.

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1,2]. We have investigated the effect of bromouridine (BrU) in pre-mRNA on the efficiency of splicing. An adenovirus major late II construct was used to prepare four different transcripts, each containing a different amount of BrU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in the transcript were substituted for BrU. Splicing was restored to some extent if 50% of the Us were replaced by BrU. The splicing efficiency returned to an almost normal level if only 1 our of every 10 Us was substituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally in vitro. Furthermore, these results strongly suggest that some Us in the adenoviral transcript, probably those at the splice sites, cannot be replaced by BrU and are therefore critical in the splicing reaction.

Vestigial suppressor genes and resistance to aminopterin in Drosophila melanogaster.

We have shown that the vestigial (vg) mutant of D. melanogaster has a perturbed nucleotide metabolism compared to various wild-type strains. The mutant is particularly spontaneously resistant to aminopterin. The resistance seems to correlate with an increase in dihydrofolate reductase (DHFR) activity and quantity. The DHRF is a target enzyme of aminopterin. Our results suggest that the vg+ gene could be a regulatory gene acting on the DHFR gene. The wing mutant phenotype being due to a decrease in the thymidylate pool (dTMP) (Silber et al., 1989). In order to understand better the action of the mutant gene on nucleotide metabolism, we have induced suppressor genes of the mutant phenotype by mutagenesis with ethyl methanesulfonate (EMS) and bromouridine (BUR). The suppressor strains obtained display a phenotype intermediate between wild-type and vg phenotype. The action of three independent suppressor genes on eight parameters of nucleotide metabolism is reported here [three enzyme activities, resistance to aminopterin and to fluorodeoxyuridine (FUdR), auxotrophy test and the ability to use exogenous thymidine and uridine]. In comparison to the original vg strain, major changes for the parameters tested are observed. The most striking effects are obtained with the vgBUR27 strain, which is highly sensitive to aminopterin and to fluorodeoxyuridine and didplas the highest thymidine kinase (TK) and DHFR activities within the strains tested. The potential actions of suppressor genes on the vg mutant are discussed.

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.