Nonpolyadenylated Transcripts Research Articles

Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians

BackgroundSingle-cell RNA-sequencing (scRNA-seq) relies on PCR amplification to retrieve information from vanishingly small amounts of starting material. To selectively enrich mRNA from abundant non-polyadenylated transcripts, poly(A) selection is a key step during library preparation. However, some transcripts, such as mitochondrial genes, can escape this elimination and overwhelm libraries. Often, these transcripts are removed in silico, but whether physical depletion improves detection of rare transcripts in single cells is unclear.ResultsWe find that a single 16S ribosomal RNA is widely enriched in planarian scRNA-seq datasets, independent of the library preparation method. To deplete this transcript from scRNA-seq libraries, we design 30 single-guide RNAs spanning its length. To evaluate the effects of depletion, we perform a side-by-side comparison of the effects of eliminating the 16S transcript and find a substantial increase in the number of genes detected per cell, coupled with virtually complete loss of the 16S RNA. Moreover, we systematically determine that library complexity increases with a limited number of PCR cycles following CRISPR treatment. When compared to in silico depletion of 16S, physically removing it reduces dropout rates, retrieves more clusters, and reveals more differentially expressed genes.ConclusionsOur results show that abundant transcripts reduce the retrieval of informative transcripts in scRNA-seq and distort the analysis. Physical removal of these contaminants enables the detection of rare transcripts at lower sequencing depth, and also outperforms in silico depletion. Importantly, this method can be easily customized to deplete any abundant transcript from scRNA-seq libraries.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

TERRA stability is regulated by RALY and polyadenylation in a telomere-specific manner

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.

High-throughput total RNA sequencing in single cells using VASA-seq

Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

SUMMARYTransposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous “nuclear exosome targeting” (NEXT) and “human silencing hub” (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co‐evolution between hosts’ and parasites’ genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral‐like silencing response. To investigate this response’s activation, we studied the single‐copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full‐length genomic flGAG‐POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG‐POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5’OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA‐DEPENDENT‐RNA‐POLYMERASE‐6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto‐unrecognized “translation‐dependent silencing” (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid-Phase Catalysis Approach.

The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.

Abstract 2287: Detection of RNA-Seq library preparation type via random forest

Abstract Library preparation protocols for RNA sequencing (RNA-Seq) libraries vary and affect the downstream analysis of RNA-Seq data. Available RNA sequencing datasets often lack library construction protocol information in the metadata. This information is necessary to be able to compare RNA sequencing datasets appropriately. For example, non-polyadenylated transcripts are measured when a ribosomal RNA-depletion method is used (riboD library preparation), but not when transcripts are selected by poly(A) tails (polyA library preparation). Without knowing the library preparation method, it would appear that the non-polyadenylated transcripts were expressed at much higher levels in the samples prepared via riboD. In order to tackle this issue, we have developed a Random Forest classifier that can delineate between riboD and polyA RNA-Seq datasets. A grid search method was applied to the number of trees, maximum depth, and the minimum datasets included in each leaf node, which determined the best parameters to be 100, 8, and 1 respectively. However, applying the model on the Pediatric Brain Tumor Atlas (PBTA) showed strong overfitting for polyA samples. We examined the performance of the model investigating each maximum depth increment from 1 to 8, and we determined that the best performance on our validation set was achieved with maximum depth of 1. We subsequently proceeded to train our classifier on our own curated compendiums of pediatric cancer polyA and riboD samples including 188 and 264 samples respectively, after balancing the two datasets in terms of disease prevalence, and selecting the top 5,000 most variable genes as the default input dimensionality of the model. For samples whose genes do not exactly match the predetermined 5,000 genes, we substitute the expression of the missing gene with the mean expression of the gene observed in the training data. We show that it achieves 100% classification accuracy of samples to their respective library preparation protocols in GTEX (all polyA), CCLE (all polyA), and for 7 of 9 SRA projects. Five of the SRA projects contained only riboD datasets, 3 contained only all polyA datasets, and one was 50% riboD, and 50% polyA. Notably the SRA datasets are not all cancer related datasets, showing the power of our model to distinguish between library preparation protocols in vastly different settings. The model will become available on Docker so that it is readily, and easily accessible for application on new samples. Our model serves as an important step towards robust library preparation identification. Including samples in the training procedure from diverse contexts would make our model more widely applicable. Need summary statement here and/or future work statement Citation Format: Ioannis Anastopoulos, Holly Beale, Geoff Lyle, Allison Cheney, Olena M. Vaske, Joshua M. Stuart. Detection of RNA-Seq library preparation type via random forest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2287.

Reovirus Low-Density Particles Package Cellular RNA.

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

Emerging roles of centromeric RNAs in centromere formation and function.

Centromeres are specialized chromosomal domains involved in kinetochore formation and faithful chromosome segregation. Despite a high level of functional conservation, centromeres are not identified by DNA sequences, but by epigenetic means. Universally, centromeres are typically formed on highly repetitive DNA, which were previously considered to be silent. However, recent studies have shown that transcription occurs in this region, known as centromeric-derived RNAs (cenRNAs). CenRNAs that contribute to fundamental aspects of centromere function have been recently investigated in detail. However, the distribution, behavior and contributions of centromeric transcripts are still poorly understood. The aim of this article is to provide an overview of the roles of cenRNAs in centromere formation and function. We describe the structure and DNA sequence of centromere from yeast to human. In addition, we briefly introduce the roles of cenRNAs in centromere formation and function, kinetochore structure, accurate chromosome segregation, and pericentromeric heterochromatin assembly. Centromeric circular RNAs (circRNAs) and R-loops are rising stars in centromere function. CircRNAs have been successfully identified in various species with the assistance of high-throughput sequencing and novel computational approaches for non-polyadenylated RNA transcripts. Centromeric R-loops can be identified by the single-strand DNA ligation-based library preparation technique. But the molecular features and function of these centromeric R-loops and circRNAs are still being investigated. In this review, we summarize recent findings on the epigenetic regulation of cenRNAs across species, which would provide useful information about cenRNAs and interesting hints for further studies.

Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

The role of Mediator and Little Elongation Complex in transcription termination

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3′ end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

Polyadenylation of Sine Transcripts Generated by RNA Polymerase III Dramatically Prolongs Their Lifetime in Cells

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various tRNAs and less often from 5S rRNA and 7SL RNA. Similar to the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the RNA polymerase III transcripts. It is well known that this polyadenylation of many RNA polymerase II transcripts (e.g., mRNAs) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20-30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the RNA from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding RNAs, which are stable in a cell due to polyadenylation.

Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.

3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

Enhancer Rna's In Brain Function and Disease

Distal enhancer elements in DNA enable higher-order chromatin interactions that regulate gene expression programs critical for cellular development, phenotype, and function. The vast majority of genetic variants linked to human health and disease by genome-wide association studies are located in non-coding regions of the genome, with putative enhancers containing more disease-linked single-nucleotide polymorphisms than all other genetic loci combined. This is especially relevant to intellectual and neuropsychiatric disorders, as sequence variations in enhancer regions have been linked to depression, schizophrenia, bipolar disorder, and autism spectrum disorders. In neuronal systems, enhancer elements are subject to widespread, bidirectional transcription that yields non-coding enhancer RNA (eRNA). However, the precise function of eRNAs has remained controversial, with different models proposing unique regulatory functions. This presentation will examine the biogenesis, localization, and function of eRNAs arising from enhancers that regulate activity-responsive immediate early genes known to be involved in cognitive functions. We show that eRNA expression from these loci are dynamically modulated by neuronal activity in an RNA PolII-dependent manner, resulting in non-polyadenylated transcripts with exclusive localization in the cell nucleus. Enhancer RNA knockdown results in impaired mRNA expression from linked genes, whereas eRNA upregulation or targeting with CRISPR-dCas9 tools results in increased expression of linked genes. Finally, we show that eRNAs interact with key epigenetic modifiers and are correlated with specific chromatin states. Together, these results suggest that RNAs transcribed from neuronal enhancers are important contributors to epigenetic landscapes and gene regulatory mechanisms. This growing link between enhancer activity and brain function strongly highlights the need to better understand the specific interactions that regulate enhancer function at the molecular level, and also suggests that enhancers could be attractive targets for a new generation of disease therapeutics.

Single-Cell RNA-Seq by Multiple Annealing and Tailing-Based Quantitative Single-Cell RNA-Seq (MATQ-Seq).

Single-cell technologies have emerged as advanced tools to study various biological processes that demand the single cell resolution. To detect subtle heterogeneity in the transcriptome, high accuracy and sensitivity are still desired for single-cell RNA-seq. We describe here multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) with ~90% capture efficiency. In addition, MATQ-seq is a total RNA assay allowing for detection of nonpolyadenylated transcripts.

PO-307 Paradoxical decrease in H2AFX copy number yet increasing mRNA expression in human breast cancer

IntroductionHistones are basic proteins responsible for DNA packaging in eukaryotic cell nuclei. H2AX is an H2A variant that plays an important role early in the DNA damage response. Although H2AX expression has been investigated, the direct contribution of this abundant chromatin protein in breast cancer is not well understood. The H2AFX gene encodes the same H2AX protein by both a short non-polyadenylated transcript and a longer polyadenylated transcript. We are characterising H2AFX copy number (CN), mRNA expression and protein abundance in breast cancer to discover if this foundation chromatin component contributing to genome stability could play a role in breast cancer.Material and methodsH2AFX mRNA abundance was analysed in a panel of breast cell lines by absolute quantitative PCR. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset was used for bioinformatic analysis.Results and discussionsAnalysis of the METABRIC dataset shows that H2AFX copy number alterations involving loss of one allele are very common (41%) and correlate with genome instability. No diploid deletions are observed, and only 2% of samples show H2AFX CN amplifications. No mutations were observed in the H2AFX coding region. Although we observed that the H2AFX short transcript is more abundant than the long transcript in the panel of breast cell lines, we found that the ratio of short and long transcript is constant. This validates the relevance of transcriptomic databases using polyA-selected H2AFX mRNAs that only report long transcript abundance. Analysis of H2AFX mRNA levels in METABRIC revealed an increase in H2AFX expression with worsening prognosis, despite the decrease in gene dosage. Furthermore, we found that H2AFX copy number decreases while H2AFX mRNA expression increases during the progression from luminal A to luminal B which are the most common breast cancer subtypes.ConclusionAnalysis of primary breast tumour genomic datasets revealed that H2AFX frequently has reduced copy number but is almost never fully deleted, mutated, or amplified. One copy is lost usually as a result of a major deletion in the 11q chromosome arm. Paradoxically, H2AFX expression is frequently increased suggesting that an independent mechanism for regulation exists to drive up-regulation. We are now bringing together H2AFX copy number, mRNA expression and protein data to understand whether H2AFX expression responds to challenges to genome integrity in breast cancer.