VPS13A and VPS13C Influence Lipid Droplet Abundance.

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson's disease, respectively.

APEX Proximity Labeling of Stress Granule Proteins.

Ascorbate peroxidase (APEX)-catalyzed proximity labeling has been recently established as a robust approach to uncover localized protein environments and transient protein-protein interactions occurring across mammalian cells. This molecular tool enables improved identification of individual proteins localized to and involved in specific cellular and subcellular pathways and functions. Engineering of an APEX2 fusion protein into the endogenous loci of proteins of interest enables directed biotinylation of neighboring polypeptides and mRNAs.

Huntington's disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization.

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology.

Author Correction: Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells.

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas.

Phenotypic Suppression of ALS/FTD-Associated Neurodegeneration Highlights Mechanisms of Dysfunction.

A fundamental question regarding the etiology of amyotrophic lateral sclerosis (ALS) is whether the various gene mutations associated with the disease converge on a single molecular pathway or act through multiple pathways to trigger neurodegeneration. Notably, several of the genes and cellular processes implicated in ALS have also been linked to frontotemporal dementia (FTD), suggesting these two diseases share common origins with varied clinical presentations. Scientists are rapidly identifying ALS/FTD suppressors that act on conserved pathways from invertebrates to vertebrates to alleviate degeneration.

Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD.

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids.

Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics.

Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics.

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective -deficient mice.

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by , is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development.

Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules.

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs.

Experimental approaches to studying the nature and impact of splicing variation in zebrafish.

From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes.

Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S.

Minor class splicing shapes the zebrafish transcriptome during development.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown.

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls.

In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation.

Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophŏnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal.

Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome.