Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Related Articles

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Mol Cell. 2019 Apr 26;:

Authors: Ali I, Ruiz DG, Ni Z, Johnson JR, Zhang H, Li PC, Khalid MM, Conrad RJ, Guo X, Min J, Greenblatt J, Jacobson M, Krogan NJ, Ott M

Abstract
Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

PMID: 31054975 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

A Unified Resource for Tracking Anti-CRISPR Names.

Related Articles

A Unified Resource for Tracking Anti-CRISPR Names.

CRISPR J. 2018 Oct;1:304-305

Authors: Bondy-Denomy J, Davidson AR, Doudna JA, Fineran PC, Maxwell KL, Moineau S, Peng X, Sontheimer EJ, Wiedenheft B

PMID: 31021273 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

Related Articles

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

CRISPR J. 2019 Feb;2:23-30

Authors: Hwang S, Maxwell KL

Abstract
The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

PMID: 31021234 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

scClustViz - Single-cell RNAseq cluster assessment and visualization.

Read Full Article on External Site Read Full Article on External Site Related Articles

scClustViz - Single-cell RNAseq cluster assessment and visualization.

F1000Res. 2018;7:

Authors: Innes BT, Bader GD

Abstract
Single-cell RNA sequencing (scRNAseq) represents a new kind of microscope that can measure the transcriptome profiles of thousands of individual cells from complex cellular mixtures, such as in a tissue, in a single experiment. This technology is particularly valuable for characterization of tissue heterogeneity because it can be used to identify and classify all cell types in a tissue. This is generally done by clustering the data, based on the assumption that cells of a particular type share similar transcriptomes, distinct from other cell types in the tissue. However, nearly all clustering algorithms have tunable parameters which affect the number of clusters they will identify in data. The R Shiny software tool described here, scClustViz, provides a simple interactive graphical user interface for exploring scRNAseq data and assessing the biological relevance of clustering results. Given that cell types are expected to have distinct gene expression patterns, scClustViz uses differential gene expression between clusters as a metric for assessing the fit of a clustering result to the data at multiple cluster resolution levels. This helps select a clustering parameter for further analysis. scClustViz also provides interactive visualisation of: cluster-specific distributions of technical factors, such as predicted cell cycle stage and other metadata; cluster-wise gene expression statistics to simplify annotation of cell types and identification of cell type specific marker genes; and gene expression distributions over all cells and cell types. scClustViz provides an interactive interface for visualisation, assessment, and biological interpretation of cell-type classifications in scRNAseq experiments that can be easily added to existing analysis pipelines, enabling customization by bioinformaticians while enabling biologists to explore their results without the need for computational expertise. It is available at https://baderlab.github.io/scClustViz/.

PMID: 31016009 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Muted fibrosis from protected islets.

Related Articles

Muted fibrosis from protected islets.

Nat Biomed Eng. 2018 Nov;2(11):791-792

Authors: Vlahos AE, Sefton MV

PMID: 31015616 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Characterizing the protein corona of sub-10 nm nanoparticles.

Related Articles

Characterizing the protein corona of sub-10 nm nanoparticles.

J Control Release. 2019 Apr 17;:

Authors: Glancy D, Zhang Y, Wu JLY, Ouyang B, Ohta S, Chan WCW

Abstract
Studies into the interactions of serum proteins with nanoparticles are typically performed using nanoparticles that are larger than the size of proteins. Due to this size discrepancy, adsorbed proteins are commonly depicted as a globular structure surrounding a nanoparticle. Here, we asked how we should view nanoparticle-protein complexes when the nanoparticles are of similar size or smaller than the proteins with which they interact. We showed that nanoparticles can serve as a cargo on a protein rather than as a carrier of the protein in a size-dependent manner. This occurs when nanoparticles are below 10 nm. We discovered that when the nanoparticle is a cargo on the protein, the binding of the protein to the receptor target is minimally affected in contrast to the nanoparticle serving as a carrier. Our study should change how we view and describe nanoparticle-protein complexes when the nanoparticles involved are equal in size or smaller than proteins.

PMID: 31004667 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis.

Related Articles

A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis.

Nat Commun. 2019 Apr 17;10(1):1791

Authors: Chapman EM, Lant B, Ohashi Y, Yu B, Schertzberg M, Go C, Dogra D, Koskimäki J, Girard R, Li Y, Fraser AG, Awad IA, Abdelilah-Seyfried S, Gingras AC, Derry WB

Abstract
Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.

PMID: 30996251 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

Related Articles

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

EMBO J. 2019 Apr 15;:

Authors: Maslon MM, Braunschweig U, Aitken S, Mann AR, Kilanowski F, Hunter CJ, Blencowe BJ, Kornblihtt AR, Adams IR, Cáceres JF

Abstract
The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

PMID: 30988016 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector.

Related Articles

Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector.

BMC Biotechnol. 2019 Apr 15;19(1):21

Authors: Mayers S, Moço PD, Maqbool T, Silva PN, Kilkenny DM, Audet J

Abstract
BACKGROUND: A robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells.
RESULTS: Primary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation.
CONCLUSIONS: Genetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products.

PMID: 30987611 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Genetic interaction networks in cancer cells.

Related Articles

Genetic interaction networks in cancer cells.

Curr Opin Genet Dev. 2019 Apr 08;54:64-72

Authors: Mair B, Moffat J, Boone C, Andrews BJ

Abstract
The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.

PMID: 30974317 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄