scholarly journals Cell-type-specific alternative polyadenylation (APA) genes reveal the function of dynamic APA in complex tissues

2020 ◽  
Author(s):  
Yulong Bai ◽  
Yidi Qin ◽  
Zhenjiang Fan ◽  
Robert M. Morrison ◽  
KyongNyon Nam ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Cell Type ◽  
Simulation Data ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
Brain Data

ABSTRACTAlternative polyadenylation (APA) causes shortening or lengthening of the 3’-untranslated region (3’-UTR) of genes across multiple cell types. Bioinformatic tools have been developed to identify genes that are affected by APA (APA genes) in single-cell RNA-Seq (scRNA-Seq) data. However, they suffer from low power, and they cannot identify APA genes specific to each cell type (cell-type-specific APA) when multiple cell types are analyzed. To address these limitations, we developed scMAPA that systematically integrates two novel steps. First, scMAPA quantifies 3’-UTR long and short isoforms without requiring assumptions on the read density shape of input data. Second, scMAPA estimates the significance of the APA genes for each cell type while controlling confounders. In the analyses on our novel simulation data and human peripheral blood mono cellular data, scMAPA showed enhanced power in identifying APA genes. Further, in mouse brain data, scMAPA identifies cell-type-specific APA genes, improving interpretability for the cell-type-specific function of APA. We further showed that this improved interpretability helps to understand a novel role of APA on the interaction between neurons and blood vessels, which is critical to maintaining the operational condition of brains. With high sensitivity and interpretability, scMAPA shed novel insights into the function of dynamic APA in complex tissues.Key PointsWe developed a bioinformatic tool, scMAPA, that identifies dynamic APA across multiple cell types and a novel simulation pipeline to assess performance of such tools in APA calling.In simulation data of various scenarios from our novel simulation pipeline, scMAPA achieves sensitivity with a minimal loss of specificity.In human peripheral blood monocellular data, scMAPA identifies APA genes accurately and robustly, finding unique associations of APA with hematological processes.scMAPA identifies APA genes specific to each cell type in mouse brain data while controlling confounders that sheds novel insights into the complex molecular processes.

scholarly journals Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

2021 ◽  
Author(s):  
Sruti Rayaprolu ◽  
Sara Bitarafan ◽  
Ranjita Betarbet ◽  
Sydney N Sunna ◽  
Lihong Cheng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Neurological Diseases ◽  
Neuronal Cell ◽  
Cell Types ◽  
Proteomic Profiling ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific ◽  
The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

scholarly journals PolyASite 2.0: a consolidated atlas of polyadenylation sites from 3′ end sequencing

2019 ◽  
Author(s):  
Christina J Herrmann ◽  
Ralf Schmidt ◽  
Alexander Kanitz ◽  
Panu Artimo ◽  
Andreas J Gruber ◽  
...  
Keyword(s):  
Alternative Polyadenylation ◽  
Quality Measures ◽  
Cell Types ◽  
Cell Type ◽  
Transcript Isoforms ◽  
Cleavage And Polyadenylation ◽  
Integrated Processing ◽  
Cell Type Specific ◽  
Polyadenylation Sites ◽  
Polyadenylation Signals

Abstract Generated by 3′ end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between human transcript isoforms. More than a dozen protocols have been developed so far for capturing and sequencing RNA 3′ ends from a variety of cell types and species. In previous studies, we have used these data to uncover novel regulatory signals and cell type-specific isoforms. Here we present an update of the PolyASite (https://polyasite.unibas.ch) resource of poly(A) sites, constructed from publicly available human, mouse and worm 3′ end sequencing datasets by enforcing uniform quality measures, including the flagging of putative internal priming sites. Through integrated processing of all data, we identified and clustered sites that are closely spaced and share polyadenylation signals, as these are likely the result of stochastic variations in processing. For each cluster, we identified the representative - most frequently processed - site and estimated the relative use in the transcriptome across all samples. We have established a modern web portal for efficient finding, exploration and export of data. Database generation is fully automated, greatly facilitating incorporation of new datasets and the updating of underlying genome resources.

scholarly journals Transposon-mediated, cell type-specific transcription factor recording in the mouse brain

2019 ◽  
Author(s):  
Alexander J. Cammack ◽  
Arnav Moudgil ◽  
Tomas Lagunas ◽  
Michael J. Vasek ◽  
Mark Shabsovich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Mouse Tissue ◽  
Specific Cell ◽  
Cell Type ◽  
Cell Fate Decisions ◽  
Cell Type Specific ◽  
The Brain

AbstractTranscription factors (TFs) play a central role in the regulation of gene expression, controlling everything from cell fate decisions to activity dependent gene expression. However, widely-used methods for TF profiling in vivo (e.g. ChIP-seq) yield only an aggregated picture of TF binding across all cell types present within the harvested tissue; thus, it is challenging or impossible to determine how the same TF might bind different portions of the genome in different cell types, or even to identify its binding events at all in rare cell types in a complex tissue such as the brain. Here we present a versatile methodology, FLEX Calling Cards, for the mapping of TF occupancy in specific cell types from heterogenous tissues. In this method, the TF of interest is fused to a hyperactive piggyBac transposase (hypPB), and this bipartite gene is delivered, along with donor transposons, to mouse tissue via a Cre-dependent adeno-associated virus (AAV). The fusion protein is expressed in Cre-expressing cells where it inserts transposon “Calling Cards” near to TF binding sites. These transposons permanently mark TF binding events and can be mapped using high-throughput sequencing. Alternatively, unfused hypPB interacts with and records the binding of the super enhancer (SE)-associated bromodomain protein, Brd4. To demonstrate the FLEX Calling Card method, we first show that donor transposon and transposase constructs can be efficiently delivered to the postnatal day 1 (P1) mouse brain with AAV and that insertion profiles report TF occupancy. Then, using a Cre-dependent hypPB virus, we show utility of this tool in defining cell type-specific TF profiles in multiple cell types of the brain. This approach will enable important cell type-specific studies of TF-mediated gene regulation in the brain and will provide valuable insights into brain development, homeostasis, and disease.

scholarly journals A Single Cell Transcriptomic Atlas Characterizes Aging Tissues in the Mouse

2019 ◽  
Author(s):  
◽  
Angela Oliveira Pisco ◽  
Aaron McGeever ◽  
Nicholas Schaum ◽  
Jim Karkanias ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Cell Type ◽  
Cellular Processes ◽  
Progressive Loss ◽  
Age Related ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
Molecular Information ◽  
Insight Into

AbstractAging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes which underlie progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes we have created a single cell transcriptomic atlas across the life span of Mus musculus which includes data from 23 tissues and organs. We discovered cell-specific changes occurring across multiple cell types and organs, as well as age related changes in the cellular composition of different organs. Using single-cell transcriptomic data we were able to assess cell type specific manifestations of different hallmarks of aging, such as senescence3, genomic instability4 and changes in the organism’s immune system2. This Tabula Muris Senis provides a wealth of new molecular information about how the most significant hallmarks of aging are reflected in a broad range of tissues and cell types.

scholarly journals Studies on the fibronectin receptors of human peripheral blood leukocytes. Morphologic and functional characterization.

1984 ◽  
Vol 159 (1) ◽  
pp. 137-151 ◽  
Author(s):  
C G Pommier ◽  
J O'Shea ◽  
T Chused ◽  
K Yancey ◽  
M M Frank ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Specific Binding ◽  
Functional Characterization ◽  
Fluid Phase ◽  
Cell Types ◽  
Cell Type ◽  
Phagocytic Cells ◽  
Blood Leukocytes ◽  
Specific Binding Sites

We have investigated the interactions between plasma fibronectin (Fn) and human peripheral blood phagocytic cells. As shown by studies of the binding of Fn-coated fluorescent microspheres (Fn-ms), both polymorphonuclear leukocytes (PMN) and monocytes had specific binding sites for Fn at the plasma membrane. However, as purified from blood, only monocytes were stimulated by Fn to become more actively phagocytic. This increase in phagocytosis was reflected by an Fn-induced increase in the ingestion of IgG-coated erythrocytes and, more dramatically by an Fn-dependent initiation of phagocytosis of C3b-coated erythrocytes. Despite this difference between PMN and monocytes in the functional consequences of Fn binding, the cell surface molecules responsible for Fn binding on the two cell types shared many characteristics. On both cells, binding of Fn-ms was inhibited by sufficient concentrations of fluid-phase Fn; both PMN and monocytes bound fewer Fn-ms at 4 degrees C than at 37 degrees C; both achieved maximal binding at similar Fn-ms/cell ratios; and phenylmethylsulfonyl fluoride did not inhibit Fn-ms binding to either cell type. Most dramatically, monoclonal anti-Fn antibodies that inhibited binding of Fn-ms to one cell type inhibited binding to both; conversely, monoclonal anti-Fn antibodies that did not inhibit Fn-ms binding to either cell type did not inhibit binding to the other. Fn will stimulate PMN to a more actively phagocytic state, like that induced in monocytes, if the PMN are first exposed to C5a or N-formyl-methionyl-leucylphenylalanine. This effect occurs without apparent change in the number of Fn receptors. We conclude that the PMN and monocyte receptors for Fn are very similar, but that their milieu is very different in the two cells as purified from peripheral blood. Whereas Fn induces increased phagocytosis in monocytes, PMN must be activated before the Fn can be effective.

scholarly journals Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Elin Lundin ◽  
Chenglin Wu ◽  
Albin Widmark ◽  
Mikaela Behm ◽  
Jens Hjerling-Leffler ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Editing ◽  
Mouse Brain ◽  
Developmental Stages ◽  
Cell Types ◽  
Specific Cell ◽  
Cell Type ◽  
Cell Type Specific ◽  
Specific Regulation

Abstract Background Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. Results We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts. Conclusions Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

scholarly journals ‘Chromatic’ Neuronal Jamming in a Primitive Brain

2018 ◽  
Author(s):  
Margarita Khariton ◽  
Xian Kong ◽  
Jian Qin ◽  
Bo Wang
Keyword(s):  
Cell Types ◽  
Physical Contact ◽  
Specific Interactions ◽  
Cell Type ◽  
Cell Diversity ◽  
Multiple Cell ◽  
Cell Type Specific ◽  
Inanimate Matter ◽  
The Brain ◽  
Jammed State

ABSTRACTJamming models developed in inanimate matter have been widely used to describe cell packing in tissues1–7, but predominantly neglect cell diversity, despite its prevalence in biology. Most tissues, animal brains in particular, comprise a mix of many cell types, with mounting evidence suggesting that neurons can recognize their respective types as they organize in space8–11. How cell diversity revises the current jamming paradigm is unknown. Here, in the brain of the flatworm planarian Schmidtea mediterranea, which exhibits remarkable tissue plasticity within a simple, quantifiable nervous system12–16, we identify a distinct packing state, ‘chromatic’ jamming. Combining experiments with computational modeling, we show that neurons of distinct types form independent percolating networks barring any physical contact. This jammed state emerges as cell packing configurations become constrained by cell type-specific interactions and therefore may extend to describe cell packing in similarly complex tissues composed of multiple cell types.

scholarly journals Analysis of single cell data as it relates to aging and longevity

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 677-678
Author(s):  
Tanya Karagiannis ◽  
Todd Dowrey ◽  
Carlos Villacorta-Martin ◽  
George Murphy ◽  
Stefano Monti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Single Cell ◽  
Peripheral Blood ◽  
Age Groups ◽  
Cell Types ◽  
Cell Type ◽  
Younger Age ◽  
Cell Type Specific ◽  
Extreme Longevity

Abstract Age-related disability and diseases are known to be delayed in people living to 100 years or more. Changes in the immune system with age are known, including in cell type composition and gene expression differences. To further explore changes in extreme longevity subjects, we investigated peripheral blood immune system cell subpopulations across age and extreme longevity at a single cell resolution. We performed an integrative analysis of public scRNA-seq datasets to define consensus cell types of longevity and age, and classified cell types in our novel New England Centenarian Study dataset. We integrated these datasets together to investigate cell type specific differences at a composition and gene expression level. Our findings identified higher cell type diversity in extreme longevity subjects compared to younger age groups, but no significant difference among younger age groups demonstrating that overall composition differences are unique to longevity. We identified novel differences in myeloid and lymphocyte populations; Extreme longevity subjects have higher composition of CD14+ Monocytes, Natural Killer cells, and T gamma delta populations and lower composition of CD16+ Monocytes and dendritic populations. We characterized gene expression differences between extreme longevity and younger age groups and differences in aging across younger age groups. We found that extreme longevity cell type specific signatures overlapped with the aging signatures by at least 50%. We identified unique genes to extreme longevity that are enriched for pathways specific to immune activation and inflammation, suggesting a protective mechanism for centenarians through efficient activation and regulation of immune subpopulations in peripheral blood.

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