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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [hyperTRIBE]

ABSTRACT: Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence. To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada).

ORGANISM(S): Ustilago maydis

PROVIDER: GSE224485 | GEO | 2023/07/26

REPOSITORIES: GEO

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

Project description:Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins. In the biotrophic fungal plant pathogen Ustilago maydis, loss of the multi KH domain containing RBP, Khd4, causes aberrant cell morphology, disturbed hyphal growth and impaired virulence. To investigate the role of Khd4 in the polar growth of infectious hyphae, we established the RNA-editing based hyperTRIBE method to detect in vivo mRNA targets.This technique is especially suited to investigate proteins of high molecular weight or low expression that are difficult to purify (McMahon et al., 2016, Xu et al., 2018, Rahman et al., 2018). HyperTRIBE exploits the RNA editing activity of the catalytic domain of ADAR (Adenosine Deaminase Acting on RNA) from D. melanogaster that additionally carries the mutation E488Q to increase editing (Kuttan and Bass, 2012; designated Ada). We fused the codon-optimized catalytic Ada domain with the green fluorescent protein (see Materials and methods; enhanced version, designated Gfp, Clontech) to the C terminus of Khd4 (Khd4-Ada-Gfp). As a control for background editing, we used the strain expressing Ada-Gfp protein with N-terminal Kat fusion in the wildtype background (control-Ada). We then performed differential gene expression analysis to determine the pathological consequence of dysregulated Khd4-mediated mRNA regulation.

2023-07-26 | GSE224486 | GEO

HyperTRIBE Analysis for DDX41 in human leukemia cells

Project description:In HyperTRIBE analysis, a hyperactive mutant of the catalytic domain of ADAR enzyme is fused to an RNA-binding protein of interest in order to identify RNAs that are bound by the protein interest through RNA-sequencing (Rahman et al Nat Protoc. 2018). By discovery of RNAs where adenosines have been edited to iosines, the RNAs that were bound by the protein of interest and then edited by ADAR are identified genome-wide. We conducted this analyis for DDX41, an RNA Helicase protein with multiple described cellular functions, including RNA splicing and innate immune sensing. Because DDX41 mutations have been discovered in leukemia patients, this analysis was conducted in the human lyeloid leukemia cell line THP-1.

2022-06-01 | GSE178892 | GEO

Mapping and understanding the function of RNA in octopus brain

Project description:Coleoid cephalopods possess a highly complex nervous system and a rich behavioral repertoire that is unique within the invertebrates and is comparable to – but evolved independently from – the vertebrates (Shigeno et al. 2018). To explain this complexity, previous studies have implicated a unusually high level of mRNA editing in transcripts expressed in both the octopus and squid nervous system (Albertin et al. 2015; Alon et al. 2015; Liscovitch-Brauer et al. 2017). We have sequenced RNA across 18 tissues from the octopus O. vulgaris, and analyzed the extent of mRNA isoform usage as well as the expression of microRNAs in the nervous system in comparison to non-neuronal tissues.

2022-11-11 | GSE192550 | GEO

Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein

Project description:Most current methods to identify cell-specific RNA binding protein (RBP) targets require analyzing an extract, a strategy that is problematic with small amounts of material. We previously addressed this issue by developing TRIBE, a method that expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR. TRIBE performs Adenosine-to-Inosine editing on candidate RNA targets of the RBP. However, target identification is limited by the efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites than TRIBE, many of which are also edited by TRIBE but at a much lower editing frequency. The data have mechanistic implications for the enhanced editing activity of the HyperADARcd as part of a RBP fusion protein and also indicate that HyperTRIBE more faithfully recapitulates the known binding specificity of its RBP than TRIBE.

2017-08-19 | GSE102814 | GEO

HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSC and LSK]

Project description:Understanding RBPs’ molecular functions as well as their cell-type specific activity requires identification of RBPs’ direct mRNA targets. However, efforts to identify RNA targets of RNA binding proteins in stem cells have been hindered by limited cell numbers. Here we adapted the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to allow for global mapping of mRNA targets of the RBP MSI2 in rare mammalian adult stem cells. We applied this method to identify MSI2 RNA binding targets in mouse HSPCs (Lin- Sca1+ Kit+ or LSK cells) and mouse leukemic stem cells (LSCs). MSI2 fusion with the catalytic domain of the Hyperactive ADAR mutant (MSI2-ADA) or with the dead catalytic mutant (MSI2-DCD) were overexpressed for 48 hours in LSKs and LSCs, followed by RNA-seq. As ADAR converts A to I (G), the fusion leaves a "finger-print" where MSI2 binds. MSI2-DCD and empty vector controls were used to normalize the background editing. We show that despite comparable expression of the MSI2-ADA fusion protein and endogenous MSI2 in LSCs vs LSKs, MSI2 RNA binding activity (number of targets and editing frequency) in LSCs is significantly higher than that in LSKs. This elevated RNA binding activity is independent of abundancy of the target transcripts.

2020-03-11 | GSE132948 | GEO

HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [Molm13_ADA_RRM_controls]

2020-03-11 | GSE146770 | GEO

HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [MOLM-13]

2020-03-11 | GSE132896 | GEO

Chromatin accessibilty profiling of SCA1+ and SCA1- lineage traced VSMCs

Project description:VSMCs expressing SCA1 have increased proliferative capacity (Dobnikar et al, 2018; Worssam et al, 2022; Pan et al, 2020). We therefore, mapped chromatin accessibility changes using bulk ATAC-seq for SCA1+ and SCA1- lineage traced VSMCs.

2024-04-09 | GSE246646 | GEO

The Amino-Terminal Part of Human Filaggrin-2 is a Component of Cornified Envelopes

Project description:Filaggrin-2 (FLG2), a member of the S100-fused type protein family, is a 250 kDa protein mainly expressed in differentiated keratinocytes of the epidermis (Wu et al., 2009; Hsu et al., 2011). Its expression is reduced in atopic dermatitis (Pellerin et al., 2013), and heterozygous non-sense mutations of its gene are associated with a more persistent form of the disease in Afro-American populations (Margolis et al., 2014). Homozygous mutations of its gene are responsible for a rare skin disease called peeling skin syndrome type A (Bolling et al., 2018; Mohamad et al., 2018). However, the exact role of FLG2, a multi-domain protein, is not well known. Its COOH-tail exhibits anti-microbial activity (Hansmann et al., 2015), whereas its B domain is suspected to be involved in the hydration of the upper part of the epidermis, the cornified layer. Here, on the basis of immunological, global proteomics and in vitro enzymatic analysis we demonstrated that its amino-terminal domain is cross-linked to the cornified envelope, a pericellular resistant structure that replaces the plasma membrane during the last step of keratinocyte differentiation. In addition, depletion of FLG2 in reconstructed 3D human epidermis induced a marked fragility of cornified envelopes. These data suggest that FLG2 is important for proper mechanical strength of the cornified layer. They may explain the epidermal fragility observed in peeling skin syndrome type A patients.

2019-02-25 | PXD011833 | Pride

Project description:raw data UCEs minute wasps Cruaud et al. 2018

| PRJNA495844 | ENA

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