Single-molecule RNA FISH Research Articles

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites.

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1's known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

Quantitative comparison of single-cell RNA sequencing versus single-molecule RNA imaging for quantifying transcriptional noise.

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability. However, how best to quantify genome-wide noise, remains unclear. Here we utilize a small-molecule perturbation (IdU) to amplify noise and assess noise quantification from numerous scRNA-seq algorithms on human and mouse datasets, and then compare to noise quantification from single-molecule RNA FISH (smFISH) for a panel of representative genes. We find that various scRNA-seq analyses report amplified noise, without altered mean-expression levels, for ~90% of genes and that smFISH analysis verifies noise amplification for the vast majority of genes tested. Collectively, the analyses suggest that most scRNA-seq algorithms are appropriate for quantifying noise including a simple normalization approach, although all of these systematically underestimate noise compared to smFISH. From a practical standpoint, this analysis argues that IdU is a globally penetrant noise-enhancer molecule-amplifying noise without altering mean-expression levels-which could enable investigations of the physiological impacts of transcriptional noise.

Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs)in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots.

Post-transcriptional splicing can occur in a slow-moving zone around the gene

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Abstract P37: Decoding Cancer Dependency and Molecular Pathways of Long Noncoding RNAs with a Novel CRISPR-Cas13d Platform

Abstract Long noncoding RNAs (lncRNAs) constitute a significant portion of the human transcriptome, yet their precise role in tumor cells remains elusive. In our study focusing on multiple myeloma (MM), we employed the CRISPR-Cas13d endonuclease's RNA-targeting capabilities to establish an innovative functional transcriptomics screening platform. This platform facilitated the comprehensive identification of tumor-promoting lncRNAs (tp-lncRNAs) in MM cells and provided mechanistic insights. Utilizing RNA-seq data from 319 MM patients, we identified 5,304 expressed lncRNAs. Subsequently, we designed and implemented a pooled lentiviral library incorporating nine single-guide RNAs (sgRNAs) per identified lncRNA, alongside 500 non-targeting sgRNAs as negative controls and nine sgRNAs targeting established protein-coding oncogenes in MM (e.g., IRF4) as positive controls. This library was introduced at a low multiplicity of infection (MOI < 0.3) across five MM cell lines, each expressing a Cas13d-GFP fusion protein. After three weeks, deep sequencing and MAGeCK assessed sgRNA depletion/enrichment in MM cells. Our findings revealed ~600 tp-lncRNAs upon which MM cell lines demonstrated significant growth dependency, constituting around 12% of the identified lncRNA transcriptome. Notably, this surpassed the previous estimation of 3–5%, with considerable overlap across MM cell lines. The dependency score of numerous tp-lncRNAs rivaled or exceeded that of the positive control IRF4. To validate dependencies, we employed a library of 3,500 sgRNAs to target 314 tp-lncRNAs in vitro and in xenograft models. This approach was also applied to a panel of six non-MM cancer cell lines, identifying a broad tumor-dependency on >59 lncRNAs and a specific MM-dependency on a smaller subset of lncRNAs. We explored the sub-cellular localization and site-of-function of tp-lncRNAs using a Cas13d variant accumulating either in the cytosol or in the nucleus, coupled with sub-cellular RNA-seq and single-molecule RNA FISH. Confirming the functionality and nuclear enrichment of most tp-lncRNAs, we identified >20 hits providing dependency in the cytosol. Our investigation also unveiled multiple transcript variants within the lncRNA transcriptome, originating from the same lncRNA genes but differing in sequences, structures, and likely functions. Crucially, our screening platform precisely identified tp-lncRNA isoform(s) conferring dependencies. Finally, we combined short-term Cas13d screens with single-cell RNA-seq in H929 MM cells. Analysis of over 20,000 cells for gRNA and transcript expression enabled us to delineate molecular pathways linked to individual perturbations of over 30 lncRNAs in a single experiment. The modulated pathways included therapeutically relevant ones, such as the unfolded protein response or mitochondrial homeostasis, further suggesting a potential therapeutic benefit from targeting tp-lncRNAs. In summary, our novel platform offers a comprehensive delineation of tumor dependency on lncRNAs in MM, providing valuable insights to guide further mechanistic inquiries. Citation Format: Eugenio Morelli, Anil Aktas-Samur, Domenico Maisano, Claire Gao, Na Liu, Vanessa Favasuli, Marcello Turi, Pietro Folino, Liang Chen, Francesca Barello, Mariateresa Fulciniti, Annamaria Gulla, Kenneth Anderson, Mehmet Kemal Samur, Nikhil Munshi. Decoding Cancer Dependency and Molecular Pathways of Long Noncoding RNAs with a Novel CRISPR-Cas13d Platform [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P37.

Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.

Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification.

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.

Single-Cell Single-Molecule RNA-FISH Combined with Immunofluorescence and High-Speed and High-Resolution Scanning Analysis to Visualize the Reactivation of Latent HIV-1.

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.

CRISPR-Based Functional Transcriptomics Defines the Tumor-Dependency and Molecular Determinants of Long Noncoding RNAs in Multiple Myeloma

Long noncoding RNAs (lncRNAs) constitute most elements in the human transcriptome, yet we lack a comprehensive understanding of their role in tumor cells. Here, in multiple myeloma (MM), we exploited the RNA-targeting activity of the CRISPR-Cas13d endonuclease to develop a functional transcriptomics screening platform that enabled not only the transcriptome-wide identification of tumor-promoting-lncRNAs (tp-lncRNAs) in MM cells - outperforming any prior attempt - but also provided mechanistic information about their sub-cellular site-of-function, the isoform-conferring activity, and their downstream molecular pathways. Using RNA-seq data from CD138+ plasma cells from 319 clinically annotated, homogeneously treated, newly diagnosed MM patients, we first identified 5,304 lncRNAs that were expressed in ≥10% of patients, and then designed and generated a pooled lentiviral library with 9 sgRNAs per identified lncRNA, ~500 non-targeting sgRNAs as negative controls, and 9 sgRNAs targeting known protein-coding oncogenes in MM, such as IRF4, as positive controls. We infected this library at low MOI (<0.3) in five MM cell lines (AMO1, H929, KMS11, OPM2, R8226), each expressing a Cas13d-GFP fusion protein. After 3 weeks, we tested for sgRNAs that were relatively depleted or enriched in the MM cell population using deep sequencing and the MAGeCK robust rank aggregation algorithm. From this, we identified ~600 tp-lncRNAs upon which MM cell lines exhibit significant growth dependency, making up ~12% of the identified lncRNA transcriptome, significantly larger than the previous estimate of 3-5%. The tp-lncRNAs overlapped across MM cell lines: 17% were observed in all cell lines tested, 26% in four cell lines, 34% in at least three cell lines, and 53% in at least two cell lines. The dependency score of many tp-lncRNAs was on a par or even superior to the dependency score of the positive control IRF4 and, as expected, the top-scoring tp-lncRNAs included RROL, MALAT1, and NEAT1. We used a small library of 3,500 gRNAs to validate the dependency on 314 tp-lncRNAs and further validated the dependency on 12 tp-lncRNAs by individually targeting them using antisense oligonucleotides or CRISPR-Cas13d. These studies were followed by downstream transcriptomic analyses using either qRT-PCR or RNA-seq, which validated the growth phenotypes and informed on function. Interestingly, when comparing CRISPR-Cas13d to the current state-of-the-art CRISPR interference screens, we found that the CRISPR-Cas13d platform was superior, with a 4- to 30-fold increase in identifying tp-lncRNAs among 913 lncRNAs of intergenic origin in the same cell lines, and with the additional ability to identify tp-lncRNAs generated at gene loci that are antisense to protein-coding genes. We further investigated the sub-cellular localization and site-of-function of the tp-lncRNAs by using a Cas13d that accumulates either in the cytosol or in the nucleus as well as sub-cellular RNA-seq and single molecule RNA FISH. This way, while confirming that most tp-lncRNAs were functional and enriched in the cell nuclei, we also identified 20 hits providing dependency in the cytosol, including the small nucleolar RNA host genes 6 and 10. We observed that the lncRNA transcriptome investigated in this study contained several transcript variants produced from the same lncRNA genes, which differ in their sequences and likely in their structures and functions. Importantly, our screening platform was able to precisely identify tp-lncRNAs isoform(s) conferring dependencies even among many isoforms, a result that was corroborated by RNA-seq and/or qRT-PCR analysis after individually targeting the tp-lncRNA isoforms with Cas13d. Finally, from our screens, we identified and validated the MM dependency on the novel lncRNA Plasma Cell Specific RNA 1 ( PLASP-1), which is one of the most abundant lncRNAs in MM and normal plasma cells (median TPM>25), whereas it has significantly lower expression or is absent in 54 normal tissues and cell types. PLASP-1 is an un-spliced transcript that directly interacts with spliceosome proteins, as established in living cells. We are currently investigating its functional impact on the splicing machinery and the results will be presented. Overall, this novel platform comprehensively defines tumor dependency on lncRNAs in MM and provides information to guide further mechanistic investigation.

Glucocorticoids regulate the human non-coding genome

Abstract Glucocorticoids (GCs) are the most commonly used anti-inflammatory and immunosuppressive drugs. However, most of the work on this class of drugs has focused on their relationship to the coding genome. It has been demonstrated in recent years that non-coding genes can play important biological roles. Thus, we performed total RNA-seq and small-RNA-seq in 9 primary human cell types: B cells, CD4+ T cells, monocytes, neutrophils, endothelial cells, fibroblasts, myoblasts, osteoblasts, and preadipocytes, treated in vitro with the GC methylprednisolone. We found that the response of lncRNA genes to GCs appears to be strong and cell type-dependent, with hematopoietic cells being more responsive to GCs than non-hematopoietic cells. Among GC-responsive lncRNAs, the intergenic and RNA host subtypes are overrepresented, while antisense lncRNAs are underrepresented. GC regulation of miRNAs appears to be limited to those that are part of a larger transcript. We generated a global map of GC-responsive lncRNAs and microRNAs in the 9 cell types, and an interactive web application to allow exploration of our results. We identified the lncRNA WAKMAR2 as a GC-induced gene that may play a role in GC action in CD4+ T cells and monocytes. WAKMAR2 has been shown to influence the expression of inflammatory cytokines in non-hematopoietic cells. Single-molecule RNA FISH revealed that, in human monocyte-derived macrophages, WAKMAR2 transcripts are primarily cytoplasmic. Ongoing work is aimed at determining whether WAKMAR2 is in fact a GC-induced negative regulator of inflammatory responses in human primary cells.

RNA localization mechanisms transcend cell morphology.

RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single-molecule RNA FISH, we found that pyrimidine-rich motifs in the 5' UTRs of RP mRNAs were sufficient to drive basal RNA localization. Interestingly, the same motifs were also sufficient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of this motif was dependent on it being in the 5' UTR of the transcript, was abolished upon perturbation of the RNA-binding protein LARP1, and was reduced upon inhibition of kinesin-1. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs to these morphologically distinct locations. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

Bioprocessing of MIR17HG Results in Long and Short Noncoding RNAs with Targetable Tumor-Promoting Activity in Multiple Myeloma

Our recent studies demonstrated that multiple myeloma (MM) cells can become significantly addicted to and in turn therapeutically susceptible to the modulation of oncogenic noncoding RNAs (ncRNAs). Through large-scale CRISPR interference (CRISPRi) and CRISPR-Cas13d viability screens, we identified MIR17HG as a leading cell growth-dependency in 6 MM cell lines. MIR17HG is mainly known for producing the miRNA cluster miR-17-92, which includes six mature oncogenic miRNAs (-17, -18a, -19a, -20a, -19b, -92a). However, we observed that depleting MIR17HG pre-RNA with sgRNAs or antisense oligonucleotides (ASO) took >3 days to downregulate the miR-17-92 members, yet appreciable transcriptional changes were immediately produced followed by cell cycle arrest in G0-G1 in 10 MM cell lines and CD138+ cells from 13 MM patients. Moreover, antagonism of miR-17-92 using anti-miRs did not phenocopy the anti-MM activity of targeting the MIR17HG pre-RNA, and this activity was only partially rescued by ectopic expression of miR-17-92 mimics. Therefore, we reasoned that alternative transcripts generated at this locus could provide cell growth dependency. We analyzed our RNA-seq dataset of CD138+ cells from >300 MM patients and 70 MM cell lines for these transcripts and observed reads from regions of MIR17HG that are not included in miR-17-92. Using qRT-PCR and single molecule RNA FISH, we identified the expression of a nuclear-enriched, ~5k-nt long, polyA(+) lncRNA. This lncRNA, named lnc-17-92 was quickly downregulated within a few hours of exposure to ASOs targeting the MIR17HG pre-RNA. Using DROSHA-knockout MM cells, in which MIR17HG produces lnc-17-92 but not miR-17-92, we found that targeting the MIR17HG pre-RNA effectively antagonized MM cell growth. Using easy-to-manipulate cellular models (e.g., HCT-116), we observed stronger rescue by ectopic expression of lnc-17-92, as compared to miR-17-92, after targeting MIR17HG pre-RNA. Using RNA-protein pull down (RPPD) and RNA yeast-3-hybrid (Y3H) assays, we found that the transcription factor c-MYC and the epigenetic modulator WDR82 were binding partners for lnc-17-92. In fact, we found that lnc-17-92 mediated their protein-protein interaction by providing the chromatin scaffold for the assembly of a MYC-WDR82 complex at the promoter region of ACC1. This gene encodes the limiting enzyme for de novo lipogenesis, a metabolic pathway that we show is controlled by lnc-17-92 to promote MM cell growth. Using three different cellular models (P493-6, HMECMYC, and U226MYC), we found that targeting MIR17HG pre-RNA preferentially killed MYC+ cells. This could be explained both by lnc-17-92 acting as a chromatin scaffold for MYC and also the known role of miR-17-92 in fine-tuning the downstream transcriptional program of MYC. However, we also observed that lnc-17-92 protected MYC from proteasomal degradation and found that the miR-17-92 member miR-92a negatively regulated MYC protein expression via an, as yet, unknown mechanism. The concerted action of lnc-17-92 and miR-17-92 on MYC protein stability is under investigation and updates will also be presented. Finally, we explored MIR17HG as a therapeutic target, which includes targeting both its lncRNA and miRNAs. To develop clinically applicable inhibitors, we screened >80 fully phosphorothioated (PS), 2'-O-methoxyethyl (2'-MOE)-modified, lipid-conjugated ASOs that could either trigger RNase H-mediated degradation of MIR17HG pre-RNA (gapmeRs) or exert function via an RNase H-independent mechanism (blockmeRs). We identified an 18-mer tocopherol (T)-conjugated gapmeR G2-15b-T ("G") and an 18-mer tocopherol (T)-conjugated steric blocker SB9-19-T ("B"). We found they had potent anti-tumor effects both in vitro and in vivo in three pre-clinical animal models, including a clinically relevant PDX-NSG mouse model. Tumor growth inhibition ranged from 100% (regression) to 50% in these models. Inhibitors did not cause overt toxicity in the mice, as shown by blood cell count, clinical, and body weight analysis. These inhibitors are currently being developed for translation to clinical trials. Altogether, our studies characterize a novel oncogenic mechanism for MIR17HG and provide clinically applicable inhibitors.

Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains.

Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating ∼24-h circadian rhythms. Here we describe a streamlined single molecule Fluorescence In Situ Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of Clock, a core circadian transcription factor, in individual clock neurons in whole mount Drosophila adult brains. Specifically, we used ∼48 fluorescent oligonucleotide probes that can bind to an individual Clock mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect Clock mRNA spots in individual clock neurons. Our results demonstrate that the number of Clock mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4h after lights are turned on) with ∼80 molecules/neuron and trough levels at ZT16 with ∼30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in Drosophila brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms.

Unravelling the impact of aging on the human endothelial lncRNA transcriptome.

The incidence and prevalence of cardiovascular disease is highest among the elderly. There is a need to further understand the mechanisms behind endothelial cell aging in order to achieve vascular rejuvenation and minimize the onset of age-related vascular diseases. Long non-coding RNAs (lncRNAs) have been proposed to regulate numerous processes in the human genome, yet their function in vascular aging and their therapeutic potential remain largely unknown. This is primarily because the majority of studies investigating the impact of aging on lncRNA expression heavily rely on in vitro studies based on replicative senescence. Here, using a unique collection of young and aged endothelial cells isolated from native human arteries, we sought to characterize the age-related alterations in lncRNA expression profiles. We were able to detect a total of 4463 lncRNAs expressed in the human endothelium from which ∼17% (798) were altered in advanced age. One of the most affected lncRNAs in aging was the primate-specific, Prostate Cancer Associated Transcript (PCAT) 14. In our follow up analysis, using single molecule RNA FISH, we showed that PCAT14 is relatively abundant, localized almost exclusively in the nucleus of young endothelial cells, and silenced in the aged endothelium. Functionally, our studies proposed that downregulation of PCAT14 alters endothelial cell transcription profile and cell functions including endothelial cell migration, sprouting and inflammatory responses in vitro. Taken together, our data highlight that endothelial cell aging correlates with altered expression of lncRNAs, which could impair the endothelial regenerative capacity and enhance inflammatory phenotypes.

Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters

Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways.

Evolutionary divergence of Firre localization and expression.

Long noncoding RNAs (lncRNAs) are rapidly evolving and thus typically poorly conserved in their sequences. How these sequence differences affect the characteristics and potential functions of lncRNAs with shared synteny remains unclear. Here we show that the syntenically conserved lncRNA Firre displays distinct expression and localization patterns in human and mouse. Single molecule RNA FISH reveals that in a range of cell lines, mouse Firre (mFirre) is predominantly nuclear, while human FIRRE (hFIRRE) is distributed between the cytoplasm and nucleus. This localization pattern is maintained in human/mouse hybrid cells expressing both human and mouse Firre, implying that the localization of the lncRNA is species autonomous. We find that the majority of hFIRRE transcripts in the cytoplasm are comprised of isoforms that are enriched in RRD repeats. We furthermore determine that in various tissues, mFirre is more highly expressed than its human counterpart. Our data illustrate that the rapid evolution of syntenic lncRNAs can lead to variations in lncRNA localization and abundance, which in turn may result in disparate lncRNA functions even in closely related species.

Nup98-dependent transcriptional memory is established independently of transcription.

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. We combined smFISH with mathematical modeling and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. We confirmed this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.

Abstract PO-144: Functional characterization of estrogen-regulated divergent long noncoding RNAs in estrogen receptor-positive breast cancer

Abstract Introduction/background: Recent studies have shown that long noncoding RNAs (lncRNAs) could be potential biomarkers and therapeutic targets in cancer. LncRNAs are significant in regulating transcriptional, post-transcriptional, and epigenetic changes in human development. Previously, we identified ~1900 lncRNAs in breast cancer (BC) using advanced technologies including Global Run-On sequencing (GRO-seq) and subcellular fractionation RNA sequencing (RNAseq); intriguingly, many were transcribed bidirectionally, now referred to as divergent lncRNAs. Currently, little is known about their role in regulating estrogen-dependent signaling in BC. In this study, we have investigated the role of a previously annotated and characterized estrogen-regulated lncRNA known as lncRNA67. Materials and Methods: We used integrated genomics to compute expression across BC molecular subtypes. Single-molecule RNA-FISH (Fluorescence in Situ Hybridization) determined its localization in BC patient samples. The role of lncRNA67 in estrogen receptor-positive (ER+) BC cells was investigated using stably inducible lncRNA overexpressing MCF7 and T47D cell lines. Total-directional RNA-seq was performed to understand its effect on global gene expression profiles. Results: LncRNA67 showed distinct expression patterns across molecular subtypes of BC. RNA-FISH revealed nucleus and cytoplasm localization, indicating one to two copies of mature RNA are present in ER+ BC patient cells. Functional assays with reduced expression showed inhibited growth of BC cells. When lncRNA67 was overexpressed in ER+ BC cells, the result correlates with clinical outcome. Gene Set Enrichment Analysis (GSEA) showed its role in regulating estrogen-dependent transcription. Conclusion: Our molecular analyses suggest lncRNA67 plays a pivotal role in ER-dependent and -independent pathways. Taken together, our results indicate that lncRNAs are an integral component of cancer biology. Acknowledgments: S.S.G. is supported by a First-time Faculty Recruitment Award from the Cancer Prevention and Research Institute of Texas (CPRIT; RR170020). S.S.G. is also supported by a grant from Lizanell and Colbert Coldwell foundation. Citation Format: Enrique I. Ramos, Laura A. Sanchez-Michael, Melina J. Sedano, Barbara Yang, Ramesh Choudhari, Alana L. Harrison, Shrikanth S Gadad. Functional characterization of estrogen-regulated divergent long noncoding RNAs in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-144.

Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.

Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms.