The mRNA stability factor Khd4 defines a specific mRNA regulon for membrane trafficking in the pathogen Ustilago maydis
This ARC Annoteted Research Context contains the original data of the Sankaranarayanan et al. publication.
Original Publication
Sankaranarayanan S, Haag C, Petzsch P, Köhrer K, Matuszyńska A, Zarnack K, Feldbrügge M. The mRNA stability factor Khd4 defines a specific mRNA regulon for membrane trafficking in the pathogen Ustilago maydis. Proc Natl Acad Sci U S A. 2023 Aug 22;120(34):e2301731120. doi: 10.1073/pnas.2301731120. Epub 2023 Aug 17. PMID: 37590419; PMCID: PMC10450656.
https://www.pnas.org/doi/10.1073/pnas.2301731120
Table of Contents
Abstract
Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA- binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a com-prehensive transcriptome- wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase sign-aling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3′ untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.
Studies
The Studies (S) are named after the names of the chapters in the publication.
S1_Loss_of_Khd4_Causes_Defects_in_Polar_Growth_of_Infectious_Hyphae
S2_Establishing_HyperTRIBE_for_Fungal_RBPs
S3_Khd4_Interacts_with_mRNAs_Enriched_for_Its_Binding_Motif_AUACCC
S4_Loss_of_Khd4_Increases_the_Abundance_of_mRNAs_with_AUACCC_Motif_in_Their_3UTR
S5_AUACCC_in_the_3_UTR_Functions_as_a_Khd4-Dependent_mRNA_Stability_Element
S6_Khd4_Tightly_Regulates_the_Expression_of_Distinct_Targets_Involved_in_Membrane_Trafficking
S7_Loss_of_Khd4_Causes_Dysregulation_of_Membrane-Trafficking_Factors_in_khd4D_Cells
S8_Loss_of_Khd4_Causes_Defects_in_Vacuole_Formation_and_Localization
Assays
The Assays (A) folder contains the results of the individual experiments. The raw and processed data are stored in the dataset folder of the assay and the corresponding protocols are in the protocol folder.
Chapter 1: S1_Loss_of_Khd4_Causes_Defects_in_Polar_Growth_of_Infectious_Hyphae
S1_A1_microscopy_yeast // Figure S1
S1_A2_microscopy_hyphae // Figure 1
Chapter 2: S2_Establishing_HyperTRIBE_for_Fungal_RBPs
S2_A1_microscopy_Rrm4_Ada_strains_DIC+Rrm4-GFP // Figure S2C-F
S2_A2_protein_expression
// Figure S3 A
S2_A3_microscopy_Rfp_Gfp
// Figure S3 B-C
S2_A4_HyperTRIBE_Rrm4_analysis
// Figure 2C-D, Figure S4
S2_A5_HyperTRIBE_Rrm4_iCLIP
// Figure 2C-D, Figure S4
Chapter 3: S3_Khd4_Interacts_with_mRNAs_Enriched_for_Its_Binding_Motif_AUACCC
S3_A1_microscopy_Khd4-Gfp
// Figure S5 C-E
S3_A2_Khd4-Ada-strains_protein_expression
// Figure S5 F-G
S3_A3_HyperTRIBE_Khd4_workflow
// Figure 3A, Figure S6A
S3_A4_HyperTRIBE_motif_analysis
// Figure S6 B-E, Figure 3, Figure S7, Figure S8 A-D
S3_A5_GO-term // Figure S8 E, Figure S9
Chapter 4: S4_Loss_of_Khd4_Increases_the_Abundance_of_mRNAs_with_AUACCC_Motif_in_Their_3UTR
S4_A1_RNAseq_data
// Figure 4 A-C, Fig S10A
S4_A2_RNAseq_analysis_khd4D_vs_wt_hyphae // Figure 4 A-C, Fig S10A
Chapter 5: S5_AUACCC_in_the_3_UTR_Functions_as_a_Khd4-Dependent_mRNA_Stability_Element
S5_A1_microscopy_khd4-targets
// Figure S10B
S5_A2_Kat_protein_level
// Figure S10C, Figure 4E
S5_A3_Kat_mRNA_level
// Figure 4F
Chapter 6: S6_Khd4_Tightly_Regulates_the_Expression_of_Distinct_Targets_Involved_in_Membrane_Trafficking
S6_A1_RNAseq_analysis_yeast_vs_hyphae
// Figure 5 A-C, Figure 6, Figure S13 A-B
S6_A2_analysis_specific_targets
// Figure 5 A-C, Figure S11 B-D
S6_A3_Kat-strains_microscopy
// Figure 5D, Figure S11 F
S6_A4_relative_fluorescence_level
// Figure S11 G
Chapter 7: S7_Loss_of_Khd4_Causes_Dysregulation_of_Membrane-Trafficking_Factors_in_khd4D_Cells
S6_A1_RNAseq_analysis_yeast_vs_hyphae
// Figure 5 A-C, Figure 6, Figure S13 A-B
S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes
// Figure 6, Figure S13 A-B
Chapter 8: S8_Loss_of_Khd4_Causes_Defects_in_Vacuole_Formation_and_Localization
S8_A1_microscopy_FM4-64 // Figure S13 C-D
S8_A2_microscopy_CAMG // Figure 7
License
Copyright © 2023 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).