A multi-factor trafficking site on the spliceosomal remodeling enzyme, BRR2, recruits C9ORF78 to regulate alternative splicing

Abstract The complete inventory of regulatory factors in human spliceosomes remains unknown, and many flexibly bound components are not revealed in present spliceosome structures. The intrinsically unstructured C9ORF78 protein was detected in C complex spliceosomes but is not contained in present spliceosome structures. We found a tight interaction between C9ORF78 and the key spliceosome remodeling factor, BRR2, in a large-scale yeast two-hybrid screen, validated by targeted in vitro assays. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identified several additional C9ORF78 interactors in spliceosomes. High-resolution cryogenic electron microscopy structures revealed how C9ORF78 and the spliceosomal B complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 led to global alternative splicing changes, including a substantial usage of alternative NAGNAG 3’-splice sites, at least in part dependent on BRR2. Comparison of our structure to C* complex spliceosomes shows that C9ORF78 could contact several detected interactors from its BRR2 “home base”, in particular the RNA helicase PRPF22, a suggested 3’-splice site regulator. Together our data firmly establish C9ORF78 as a novel, late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for the suggested, but puzzling, roles of BRR2 during splicing catalysis and alternative splicing.

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Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism

10.1101/2021.10.20.465175 ◽

2021 ◽

Author(s):

Izabela Wojciechowska ◽

Thiya Mukherjee ◽

Patrick Knox-Brown ◽

Xueyun Hu ◽

Aashima Khosla ◽

...

Keyword(s):

Target Genes ◽

Affinity Purification ◽

Regulatory Protein ◽

Lipid Binding ◽

Epidermal Differentiation ◽

In Vitro Assays ◽

Dependent Manner ◽

Lipid Transfer ◽

Start Domain

Plant homeodomain leucine-zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. The START domain is required for TF activity; however, its presumed role as a lipid sensor is not well understood. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative family member which controls epidermal differentiation, recruits lysophosphatidylcholines in a START-dependent manner. In vitro assays with recombinant protein verified that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally uncovered that PDF2 controls the expression of phospholipid-related target genes by binding to a palindromic octamer with consensus to a phosphate (Pi) response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments further revealed a role for START in maintaining protein levels, and Pi limitation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Overall our data provide insights towards understanding how the lipid metabolome integrates Pi availability with gene expression.

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Snail Driving Alternative Splicing of CD44 by ESRP1 Enhances Invasion and Migration in Epithelial Ovarian Cancer

Cellular Physiology and Biochemistry ◽

10.1159/000484458 ◽

2017 ◽

Vol 43 (6) ◽

pp. 2489-2504 ◽

Cited By ~ 16

Author(s):

Le Chen ◽

Ying Yao ◽

Lijuan Sun ◽

Jiajia Zhou ◽

Minmin Miao ◽

...

Keyword(s):

Ovarian Cancer ◽

Alternative Splicing ◽

Epithelial Ovarian Cancer ◽

Epithelial Mesenchymal Transition ◽

Regulatory Protein ◽

Cell Counting ◽

Migration And Invasion ◽

Mesenchymal Transition

Background/Aims: Our study aims to investigate the role, effect and mechanisms of ESRP1 (epithelial splicing regulatory protein 1) in epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (EOC). Methods: Microarray and immunohistochemical analysis of ESRP1 expression were performed in EOC cases. The correlations between ESRP1 expression and clinical factors on EOC were assessed. Lentivirus-mediated RNA interference and EGFP vector which contains ESRP1 gene were used to down-regulate and up-regulate ESRP1 expression in human EOC cell lines. Roles of ESRP1 in cell growth, migration and invasion of EOC cells were also measured by Cell Counting Kit-8 and Transwell systems in vitro and by a nude mice intraperitoneal transplantation model in vivo. Results: By the analysis of Gene Expression Omnibus (GEO) (p<0.05) and our own microarray data (p<0.001), ESRP1 expression in EOC was significantly different from normal ovarian tissue. It was abundant in the nuclei of cancer cells and in malignant lesions. However, it was weakly expressed or negative in both normal and benign lesions. High ESRP1 expression in EOC was associated with poor clinical outcomes. Decreased ESRP1 expression significantly increased cell migration and invasion both in vivo and in vitro. Snail strongly repressed ESRP1 transcription through binding to the ESRP1 promoter in EOC cells. Furthermore, ESRP1 regulated the expression of CD44s. Down-regulated ESRP1 resulted in an isoform switching from CD44v to CD44s, which modulated epithelial-mesenchymal transition (EMT) program in EOC. Up-regulatin of ESRP1 was detected in mesenchymal to epithelial transition (MET) in vivo. Conclusions: ESRP1 regulates CD44 alternative splicing during the EMT process which plays an important role in EOC carcinogenesis. In addition, ESRP1 is associated with disease prognosis in EOC.

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Detailed characterization of the substrate specificity of mouse wax synthase.

Acta Biochimica Polonica ◽

10.18388/abp.2013_1973 ◽

2013 ◽

Vol 60 (2) ◽

Cited By ~ 8

Author(s):

Magdalena Miklaszewska ◽

Adam Kawiński ◽

Antoni Banaś

Keyword(s):

Substrate Specificity ◽

Large Scale ◽

Fatty Acyl ◽

Wax Esters ◽

In Vitro Assays ◽

Esterification Reaction ◽

Scale Production ◽

Acyltransferase Activity ◽

Wax Synthase

Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

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Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes

10.2337/figshare.17026610 ◽

2021 ◽

Author(s):

Morgan J. Smith ◽

Lucia Pastor ◽

Jeremy R.B. Newman ◽

Patrick Concannon

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Alternative Splicing ◽

Cell Surface ◽

Regulatory Protein ◽

Jurkat Cells ◽

Causative Gene ◽

Transcript Isoforms

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a non-synonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling and had significantly increased levels of cell surface CD47. In primary CD4+ and CD8+ T cells cell surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.

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Large-scale identification of RBP-RNA interactions by RAPseq refines essentials of post-transcriptional gene regulation

10.1101/2021.11.08.467743 ◽

2021 ◽

Author(s):

Ionut Atanasoai ◽

Sofia Papavasileiou ◽

Natalie Preiss ◽

Claudia Kutter

Keyword(s):

Binding Sites ◽

Large Scale ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification ◽

Binding Motif ◽

The Past ◽

Optimal Distance ◽

Transcriptional Gene Regulation

Over the past decade, thousands of putative human RNA binding proteins (RBPs) have been identified and increased the demand for specifying RNA binding capacities. Here, we developed RNA affinity purification followed by sequencing (RAPseq) that enables in vitro large-scale profiling of RBP binding to native RNAs. First, by employing RAPseq, we found that vertebrate HURs recognize a conserved RNA binding motif and bind predominantly to introns in zebrafish compared to 3'UTRs in human RNAs. Second, our dual RBP assays (co-RAPseq) uncovered cooperative RNA binding of HUR and PTBP1 within an optimal distance of 27 nucleotides. Third, we developed T7-RAPseq to discern m6A-dependent and -independent RNA binding sites of YTHDF1. Fourth, RAPseq of 26 novel non-canonical RBPs revealed specialized moonlighting interactions. Last, five pathological IGF2BP family variants exhibited different RNA binding patterns. Overall, our simple, scalable and versatile method enables to fast-forward RBP-related questions.

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A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects

ICES Journal of Marine Science ◽

10.1093/icesjms/fsp194 ◽

2009 ◽

Vol 66 (10) ◽

pp. 2089-2105 ◽

Cited By ~ 18

Author(s):

Cor A. Schipper ◽

Joost Lahr ◽

Paul J. van den Brink ◽

Steve G. George ◽

Peter-Diedrich Hansen ◽

...

Keyword(s):

Retrospective Analysis ◽

Large Scale ◽

Biological Effects ◽

In Vitro Assays ◽

Multivariate Statistical ◽

Large Scale Field ◽

Hepatic Metallothionein ◽

Fish Biomarkers

Abstract Schipper, C. A., Lahr, J., van den Brink, P. J., George, S. G., Hansen, P-D., da Silva de Assis, H. C., van der Oost, R., Thain, J. E., Livingstone, D., Mitchelmore, C., van Schooten, F-J., Ariese, F., Murk, A. J., Grinwis, G. C. M., Klamer, H., Kater, B. J., Postma, J. F., van der Werf, B., and Vethaak, A. D. 2009. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. – ICES Journal of Marine Science, 66: 2089–2105. Biological-effects monitoring in estuarine environments is complex as a result of strong gradients and fluctuations in salinity and other environmental conditions, which may influence contaminant bioavailability and the physiology and metabolism of the organisms. To select the most robust and reliable biological-effect methods for monitoring and assessment programmes, a large-scale field study was conducted in two estuarine transects in the Netherlands. The locations ranged from heavily polluted harbour areas (the ports of Rotterdam and Amsterdam) to cleaner coastal and freshwater sites. Assessment methods used included a variety of biomarkers in flounder (Platichthys flesus) and a range of in vitro (sediment extracts) and in vivo bioassays. Multivariate statistical analysis was applied to investigate correlations and relationships between various biological effects and contaminant levels in flounder liver or sediments. Several biological methods seemed to be too much affected by salinity differences for routine use in estuaries. The most discriminative biomarkers in the study were hepatic metallothionein content and biliary 1-OH pyrene in fish. Mechanism-based in vitro assays DR-CALUX and ER-CALUX applied to sediment extracts for screening of potential toxicity were much more responsive than in vivo bioassays with macro-invertebrates using survival as an endpoint.

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An integrative drug repositioning framework discovered a potential therapeutic agent targeting COVID-19

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00568-6 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Yiyue Ge ◽

Tingzhong Tian ◽

Suling Huang ◽

Fangping Wan ◽

Jingxin Li ◽

...

Keyword(s):

Large Scale ◽

Therapeutic Agent ◽

Drug Repositioning ◽

In Vitro Assays ◽

In Silico Screening ◽

N Protein ◽

Drug Candidates ◽

Global Spread ◽

Wet Lab

AbstractThe global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires an urgent need to find effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). In this study, we developed an integrative drug repositioning framework, which fully takes advantage of machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 can interact with the nucleocapsid (N) protein of SARS-CoV-2 and is able to suppress the LPS-induced production of several inflammatory cytokines that are highly relevant to the prevention of immunopathology induced by SARS-CoV-2 infection.

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A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

Molecular Cancer ◽

10.1186/s12943-021-01368-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jeong A Bae ◽

Woo Kyun Bae ◽

Sung Jin Kim ◽

Yoo-Seung Ko ◽

Keon Young Kim ◽

...

Keyword(s):

Colorectal Cancer ◽

Liver Metastasis ◽

Distant Metastasis ◽

Regulatory Protein ◽

Binding Pocket ◽

In Vitro Assays ◽

Docking Model ◽

Small Molecule Compound

Abstract Background Distant metastasis is the major cause of death in patients with colorectal cancer (CRC). Previously, we identified KITENIN as a metastasis-enhancing gene and suggested that the oncogenic KITENIN complex is involved in metastatic dissemination of KITENIN-overexpressing CRC cells. Here, we attempted to find substances targeting the KITENIN complex and test their ability to suppress distant metastasis of CRC. Methods We screened a small-molecule compound library to find candidate substances suppressing the KITENIN complex in CRC cells. We selected a candidate compound and examined its effects on the KITENIN complex and distant metastasis through in vitro assays, a molecular docking model, and in vivo tumor models. Results Among several compounds, we identified DKC1125 (Disintegrator of KITENIN Complex #1125) as the best candidate. DKC1125 specifically suppressed KITENIN gain of function. After binding KH-type splicing regulatory protein (KSRP), DKC1125 degraded KITENIN and Dvl2 by recruiting RACK1 and miRNA-124, leading to the disintegration of the functional KITENIN–KSRP–RACK1–Dvl2 complex. A computer docking model suggested that DKC1125 specifically interacted with the binding pocket of the fourth KH-domain of KSRP. KITENIN-overexpressing CRC cells deregulated certain microRNAs and were resistant to 5-fluorouracil, oxaliplatin, and cetuximab. DKC1125 restored sensitivity to these drugs by normalizing expression of the deregulated microRNAs, including miRNA-124. DKC1125 effectively suppressed colorectal liver metastasis in a mouse model. Interestingly, the combination of DKC1125 with 5-fluorouracil suppressed metastasis more effectively than either drug alone. Conclusion DKC1125 targets the KITENIN complex and could therefore be used as a novel therapeutic to suppress liver metastasis in CRC expressing high levels of KITENIN.

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In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

Frontiers in Physiology ◽

10.3389/fphys.2021.697270 ◽

2021 ◽

Vol 12 ◽

Author(s):

Giorgia Palano ◽

Ariana Foinquinos ◽

Erik Müllers

Keyword(s):

Image Analysis ◽

Drug Discovery ◽

High Throughput Screening ◽

Large Scale ◽

Cardiac Fibrosis ◽

3D Models ◽

In Vitro Assays ◽

Stress Injury

As a result of stress, injury, or aging, cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components resulting in pathological remodeling, tissue stiffening, ventricular dilatation, and cardiac dysfunction that contribute to heart failure (HF) and eventually death. Currently, there are no effective therapies specifically targeting cardiac fibrosis, partially due to limited understanding of the pathological mechanisms and the lack of predictive in vitro models for high-throughput screening of antifibrotic compounds. The use of more relevant cell models, three-dimensional (3D) models, and coculture systems, together with high-content imaging (HCI) and machine learning (ML)-based image analysis, is expected to improve predictivity and throughput of in vitro models for cardiac fibrosis. In this review, we present an overview of available in vitro assays for cardiac fibrosis. We highlight the potential of more physiological 3D cardiac organoids and coculture systems and discuss HCI and automated artificial intelligence (AI)-based image analysis as key methods able to capture the complexity of cardiac fibrosis in vitro. As 3D and coculture models will soon be sufficiently mature for application in large-scale preclinical drug discovery, we expect the combination of more relevant models and high-content analysis to greatly increase translation from in vitro to in vivo models and facilitate the discovery of novel targets and drugs against cardiac fibrosis.

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