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1.
bioRxiv ; 2023 Oct 11.
Article in English | MEDLINE | ID: mdl-37873291

ABSTRACT

PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.

2.
Anal Chem ; 94(51): 17868-17876, 2022 12 27.
Article in English | MEDLINE | ID: mdl-36508568

ABSTRACT

Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.


Subject(s)
DNA , Noninvasive Prenatal Testing , Pregnancy , Female , Humans , Reproducibility of Results , DNA/chemistry , Polymerase Chain Reaction/methods , DNA Replication
3.
Elife ; 92020 08 10.
Article in English | MEDLINE | ID: mdl-32773035

ABSTRACT

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo.


Subject(s)
Nonsense Mediated mRNA Decay/physiology , Olfactory Receptor Neurons/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Animals , Cells, Cultured , Male , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA-Seq , Receptors, Odorant/genetics , Receptors, Odorant/physiology , Single-Cell Analysis
5.
Adv Exp Med Biol ; 1129: 63-79, 2019.
Article in English | MEDLINE | ID: mdl-30968361

ABSTRACT

In this review, we describe the BD Rhapsody™ Single-Cell Analysis System, a platform that allows high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and a multitier barcoding system. The resulting captured information can be used to generate various types of next-generation sequencing (NGS) libraries, including whole transcriptome analysis for discovery biology and targeted RNA analysis for high sensitivity transcript detection. The BD Rhapsody system can be used with emerging applications, such as BD™ AbSeq assays, to profile gene expression in both mRNA and protein level to provide ultra-high resolution analysis of single cells.


Subject(s)
Gene Expression Profiling , High-Throughput Nucleotide Sequencing , RNA, Messenger/analysis , Single-Cell Analysis/instrumentation , Single-Cell Analysis/methods , Sequence Analysis, RNA , Transcriptome
6.
EMBO Rep ; 20(2)2019 02.
Article in English | MEDLINE | ID: mdl-30573526

ABSTRACT

Testis-expressed X-linked genes typically evolve rapidly. Here, we report on a testis-expressed X-linked microRNA (miRNA) cluster that despite rapid alterations in sequence has retained its position in the Fragile-X region of the X chromosome in placental mammals. Surprisingly, the miRNAs encoded by this cluster (Fx-mir) have a predilection for targeting the immediately adjacent gene, Fmr1, an unexpected finding given that miRNAs usually act in trans, not in cis Robust repression of Fmr1 is conferred by combinations of Fx-mir miRNAs induced in Sertoli cells (SCs) during postnatal development when they terminate proliferation. Physiological significance is suggested by the finding that FMRP, the protein product of Fmr1, is downregulated when Fx-mir miRNAs are induced, and that FMRP loss causes SC hyperproliferation and spermatogenic defects. Fx-mir miRNAs not only regulate the expression of FMRP, but also regulate the expression of eIF4E and CYFIP1, which together with FMRP form a translational regulatory complex. Our results support a model in which Fx-mir family members act cooperatively to regulate the translation of batteries of mRNAs in a developmentally regulated manner in SCs.


Subject(s)
Fragile X Mental Retardation Protein/genetics , MicroRNAs/genetics , Multigene Family , RNA Interference , RNA, Messenger/genetics , Spermatogenesis/genetics , 3' Untranslated Regions , Animals , Gene Expression Regulation , Humans , Male , Mice , Testis/metabolism
7.
Cell Rep ; 17(1): 149-164, 2016 09 27.
Article in English | MEDLINE | ID: mdl-27681428

ABSTRACT

The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.


Subject(s)
Adult Germline Stem Cells/metabolism , Gene Expression Regulation, Developmental , Genes, X-Linked , Homeodomain Proteins/genetics , Spermatogenesis/genetics , Spermatogonia/metabolism , Adult Germline Stem Cells/cytology , Animals , Genes, Developmental , Homeodomain Proteins/metabolism , Male , Mice , Mice, Knockout , Multigene Family , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Spermatogonia/cytology
8.
Stem Cell Reports ; 6(6): 844-857, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27304915

ABSTRACT

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-ß and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.


Subject(s)
Cell Lineage/genetics , Endoderm/metabolism , Human Embryonic Stem Cells/metabolism , Nonsense Mediated mRNA Decay , Pluripotent Stem Cells/metabolism , RNA, Messenger/genetics , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Cell Differentiation , Cell Line , Ectoderm/cytology , Ectoderm/metabolism , Endoderm/cytology , Gene Expression Profiling , Human Embryonic Stem Cells/cytology , Humans , Mesoderm/cytology , Mesoderm/metabolism , Pluripotent Stem Cells/cytology , RNA Helicases , RNA, Messenger/metabolism , Sequence Analysis, RNA , Signal Transduction , Trans-Activators , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism
9.
Cell ; 165(2): 382-95, 2016 04 07.
Article in English | MEDLINE | ID: mdl-27040500

ABSTRACT

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events.


Subject(s)
Embryonic Development , Genes, Duplicate , Nonsense Mediated mRNA Decay , RNA-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Evolution, Molecular , Gametogenesis , HeLa Cells , Humans , Mice
11.
Nucleic Acids Res ; 43(19): 9314-26, 2015 Oct 30.
Article in English | MEDLINE | ID: mdl-25908788

ABSTRACT

Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in olfaction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggesting that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dramatically higher average AU-content and lower predicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU-rich elements (AREs) in their 3'UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3' UTR regions and with fewer predicted miRNA-binding sites. All of these novel properties correlated with higher Olfr expression. We also identified striking differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a finding consistent with their independent evolutionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this decay in neural lineage cells.


Subject(s)
Olfactory Mucosa/metabolism , RNA, Messenger/chemistry , Receptors, Odorant/genetics , 3' Untranslated Regions , 5' Untranslated Regions , Animals , Binding Sites , Cell Line , Female , Gene Expression Profiling , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Multigene Family , Open Reading Frames , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/metabolism , Receptors, Odorant/metabolism , Sequence Analysis, RNA
12.
Cell Rep ; 6(4): 748-64, 2014 Feb 27.
Article in English | MEDLINE | ID: mdl-24529710

ABSTRACT

The mechanisms dictating whether a cell proliferates or differentiates have undergone intense scrutiny, but they remain poorly understood. Here, we report that UPF1, a central component in the nonsense-mediated RNA decay (NMD) pathway, plays a key role in this decision by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGF-ß inhibitor SMAD7 and stimulating TGF-ß signaling. UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Neural differentiation is triggered when NMD is downregulated by neurally expressed microRNAs (miRNAs). This UPF1-miRNA circuitry is highly conserved and harbors negative feedback loops that act as a molecular switch. Our results suggest that the NMD pathway collaborates with the TGF-ß signaling pathway to lock in the stem-like state, a cellular state that is stably reversed when neural differentiation signals that induce NMD-repressive miRNAs are received.


Subject(s)
Gene Expression Regulation, Developmental , Neural Stem Cells/metabolism , Neurogenesis , Nonsense Mediated mRNA Decay , Animals , Base Sequence , Cell Line, Tumor , Cells, Cultured , Feedback, Physiological , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Sequence Data , Neural Stem Cells/cytology , Smad7 Protein/genetics , Smad7 Protein/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transforming Growth Factor beta/metabolism , Xenopus
13.
Mol Cell ; 45(3): 271-3, 2012 Feb 10.
Article in English | MEDLINE | ID: mdl-22325347

ABSTRACT

Capped and polyadenylated long noncoding RNAs (lncRNAs) are shown to be degraded by a DCP2-mediated turnover mechanism by Geisler et al. (2012); this provides a new level of regulatory control for inducible genes by lncRNAs.


Subject(s)
Exoribonucleases/genetics , Gene Expression Regulation, Fungal , RNA Caps/metabolism , RNA Processing, Post-Transcriptional , RNA, Untranslated/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics
14.
Mol Cell ; 43(6): 950-61, 2011 Sep 16.
Article in English | MEDLINE | ID: mdl-21925383

ABSTRACT

Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.


Subject(s)
RNA Stability , RNA, Messenger/metabolism , Activating Transcription Factor 3/metabolism , Blotting, Western , Feedback, Physiological , Gene Expression Regulation , HeLa Cells , Homeostasis , Humans , RNA Helicases , RNA Interference , Trans-Activators/antagonists & inhibitors
15.
Mol Cell ; 42(4): 500-10, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21596314

ABSTRACT

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.


Subject(s)
Brain/growth & development , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , RNA Stability , Trans-Activators/metabolism , Transcriptional Activation , Animals , Brain/metabolism , Chick Embryo , Exons , HEK293 Cells , HeLa Cells , Humans , Mice , MicroRNAs/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neurogenesis/genetics , Neurons/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , RNA Helicases , RNA-Binding Proteins , Rats , Trans-Activators/genetics , Xenopus laevis
16.
Proc Natl Acad Sci U S A ; 106(28): 11776-81, 2009 Jul 14.
Article in English | MEDLINE | ID: mdl-19561302

ABSTRACT

For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel.


Subject(s)
Chloride Channels/metabolism , Membrane Proteins/metabolism , Signal Transduction/physiology , Smell/physiology , Animals , Anoctamins , Cell Line , Cilia/metabolism , Humans , Mice , Olfactory Receptor Neurons/metabolism , Patch-Clamp Techniques , Proteomics
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