# The mRNA stability factor Khd4 defines a specific mRNA regulon for membrane trafficking in the pathogen Ustilago maydis This ARC [Annoteted Research Context](https://arc-rdm.org/) 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 1. [Abstract](## Abstract) 2. [Studies](## Studies) 3. [Assays](## Assays) 4. [Licence](## License) ## 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)](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/studies) 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)](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays) 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](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S1_A1_microscopy_yeast?ref_type=heads) Figure S1 [S1_A2_microscopy_hyphae](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S1_A2_microscopy_hyphae?ref_type=heads) Figure 1 ### Chapter 2: S2_Establishing_HyperTRIBE_for_Fungal_RBPs [S2_A1_microscopy_Rrm4_Ada_strains_DIC+Rrm4-GFP](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S2_A1_microscopy_Rrm4-Ada_Rrm4-Gfp) Figure S2C-F [S2_A2_protein_expression ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S2_A2_protein_expression) Figure S3 A [S2_A3_microscopy_Rfp_Gfp ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S2_A3_microscopy_Rfp_Gfp) Figure S3 B-C [S2_A4_HyperTRIBE_Rrm4_analysis ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S2_A4_HyperTRIBE_Rrm4_analysis) Figure 2C, Figure S4 A-D [S2_A5_HyperTRIBE_Rrm4_iCLIP ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S2_A5_HyperTRIBE_Rrm4_iCLIP) Figure 2D, Figure S4 E-G ### Chapter 3: S3_Khd4_Interacts_with_mRNAs_Enriched_for_Its_Binding_Motif_AUACCC [S3_A1_microscopy_Khd4-Gfp ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S3_A1_microscopy_Khd4-Gfp) Figure S5 C-E [S3_A2_Khd4-Ada-strains_protein_expression ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S3_A2_Khd4-Ada-strains_protein_expression) Figure S5 F-G [S3_A3_HyperTRIBE_Khd4_workflow ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S3_A3_HyperTRIBE_Khd4_workflow) Figure 3A, Figure S6A [S3_A4_HyperTRIBE_motif_analysis ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S3_A4_HyperTRIBE_motif_analysis) Figure S6 B-E, Figure 3, Figure S7, Figure S8 A-D [S3_A5_GO-term](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/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 ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S4_A1_RNAseq_data) Figure 4 A-C, Fig S10A [S4_A2_RNAseq_analysis_khd4D_vs_wt_hyphae](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/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 ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S5_A1_microscopy_khd4-targets) Figure S10B [S5_A2_Kat_protein_level ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S5_A2_Kat_protein_level?ref_type=heads) Figure S10C, Figure 4E [S5_A3_Kat_mRNA_level ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S5_A3_Kat_mRNA_level?ref_type=heads) Figure 4F ### Chapter 6: S6_Khd4_Tightly_Regulates_the_Expression_of_Distinct_Targets_Involved_in_Membrane_Trafficking [S6_A1_RNAseq_analysis_yeast_vs_hyphae ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae) Figure 5 A-C, Figure 6, Figure S13 A-B [S6_A2_analysis_specific_targets ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S6_A2_analysis_specific_targets) Figure 11 B-D [S6_A3_Kat-strains_microscopy ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S6_A3_Kat-strains_microscopy) Figure 5D, Figure S11 F [S6_A4_relative_fluorescence_level ](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S6_A4_relative_fluorescence_level) Figure S11 G ### Chapter 7: S7_Loss_of_Khd4_Causes_Dysregulation_of_Membrane-Trafficking_Factors_in_khd4D_Cells see S6_A1 and S6_A2 ### Chapter 8: S8_Loss_of_Khd4_Causes_Defects_in_Vacuole_Formation_and_Localization [S8_A1_microscopy_FM4-64](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S8_A1_microscopy_FM4-64?ref_type=heads) Figure S13 C-D [S8_A2_microscopy_CAMG](https://git.nfdi4plants.org/sankaran/Sankaranarayanan-2023/-/tree/master/assays/S8_A2_microscopy_CMAG?ref_type=heads) 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).