diff --git a/assays/S2_A1_microscopy_Rrm4-Ada_Rrm4-Gfp/protocols/imaging .md b/assays/S2_A1_microscopy_Rrm4-Ada_Rrm4-Gfp/protocols/imaging .md
new file mode 100644
index 0000000000000000000000000000000000000000..942f94ba2e7b4b3e5f834c37a8346d56044e294d
--- /dev/null
+++ b/assays/S2_A1_microscopy_Rrm4-Ada_Rrm4-Gfp/protocols/imaging .md	
@@ -0,0 +1 @@
+For microscopic analysis, 20 ml of yeast cells were grown in CM medium (1% glucose) to an OD600 of 0.5. Hyphal cells were induced by shifting 20 ml cell culture from CM medium to NM medium (supplemented either with 1% glucose or 1% arabinose) for 6, 9, 10, and 12 hours. All images and videos were acquired using laser-based epifluorescence-microscopy, Zeiss Axio Observer.Z1 (Oberkochen, Germany) as described previously (35).
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diff --git a/assays/S2_A2_protein_expression/protocols/Cell disruption and immunoblotting analysis.md b/assays/S2_A2_protein_expression/protocols/Cell disruption and immunoblotting analysis.md
new file mode 100644
index 0000000000000000000000000000000000000000..bae610d3996314584ac552b40ef8fe45b051069d
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+++ b/assays/S2_A2_protein_expression/protocols/Cell disruption and immunoblotting analysis.md	
@@ -0,0 +1,2 @@
+U. maydis cell disruption was performed as previously reported (32). In short, 50 ml of hyphal cells (6 h.p.i.; see Plasmids, strains, and growth conditions) were harvested by centrifugation at 7546 g, for 10 min. After washing in phosphate-buffered saline of pH 7.0, the samples were flash-frozen in liquid nitrogen and stored at -80 °C until further use. Samples were lysed using a 5 mm stainless-steel bead in Mixer Mill MM400 (Retsch, Haan, Germany) at 30 Hz for 1 min. Cell disruption was repeated 3 times with intermittent liquid cooling steps of 5 min. The resulting homogenized cell powder was resuspended in 1 ml urea buffer (8 M urea; 50 mM Tris/HCl pH 8; containing 1 mM DTT, 0.1 M PMSF, 1 tablet of cOmplete protease inhibitor per 25 ml, Roche, Mannheim, Germany) and centrifuged at 16,000 g for 10 min at 4 °C. The supernatant was used for subsequent analysis. Protein concentrations were measured with the Bradford assay (BioRad, Munich, Germany) as per the manufacturer’s protocol. Sample volumes were adjusted to equal concentrations, supplemented with Laemmli buffer, and boiled at 95 °C for 10 min. Samples were resolved in 1.5 mm thick 8% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Protran) for immobilization by semi-dry blotting.
+Proteins were detected using the primary antibodies, α-Gfp (Roche, Germany) and α-Actin from mouse (MP Biomedicals, Germany). α-mouse IgG HRP conjugate (Promega, Madison, WI, United States) was used as a secondary antibody. Antibodies bound to nitrocellulose membrane were removed by treating the blot in TBS buffer pH 3.0 (50 mM Tris pH 3.0, 150 mM NaCl) at room temperature, before detecting the constitutively expressed actin control. Detection was carried out using ECL™ Prime (Cytiva RPN2236;). Images were recorded by a luminescence image analyzer, LAS4000 (GE Healthcare).
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diff --git a/assays/S2_A4_HyperTRIBE_Rrm4_analysis/protocols/HyperTRIBE experiment and data processing.md b/assays/S2_A4_HyperTRIBE_Rrm4_analysis/protocols/HyperTRIBE experiment and data processing.md
new file mode 100644
index 0000000000000000000000000000000000000000..c4d5dda5c32145862e1f41fd9a2b48c3da4175be
--- /dev/null
+++ b/assays/S2_A4_HyperTRIBE_Rrm4_analysis/protocols/HyperTRIBE experiment and data processing.md	
@@ -0,0 +1,7 @@
+HyperTRIBE strains were grown consistently under arabinose-induced promoter-on conditions. Hyphae were induced by shifting 50 ml of exponentially growing cells from CM to NM medium, each supplemented with 1% arabinose. After 6 hours, the hyphal cells were harvested by centrifugation at 7546 g for 10 min at 4 °C. Cell pellets were resuspended in 1 ml ice-cold nuclease-free water and transferred to a 2 ml centrifuge tube. Cells were harvested at 7546 g, for 10 min at 4 °C, and the supernatant was removed completely. The resulting cell pellets were flash-frozen in liquid nitrogen and stored at -80 °C until further use.
+
+Total RNA was extracted using the RNeasy Plant Mini Kit (74904; Qiagen, Hilden, Germany) following the manufacturer’s instructions. Cell pellets were mechanically lysed at 30 Hz for 5 min at 4 °C in Mixer Mill MM400 (Retsch, Haan, Germany), in the presence of glass beads and Buffer RLC (+ß-mercaptoethanol). The resulting cell lysate was transferred to the QIAshredder spin column for further homogenization. The supernatant was transferred to a new centrifuge tube, mixed with 1 volume of 70% ethanol, and added to the RNeasy spin column. The subsequent steps were performed as per the manufacturer’s protocol. To eliminate genomic DNA contamination, the on-column DNase digestion was performed using the RNase-Free DNase set (79254; Qiagen, Hilden, Germany). VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-01/02; Vazyme) was used for cDNA library generation. All cDNA libraries expressing hyperTRIBE constructs sequenced using the HiSeq 3000 platform (Illumina), were processed simultaneously to obtain 151 nt single-end reads.
+The bioinformatics analyses were based on the U. maydis 521 genome sequence (Ustilago_maydis.Umaydis521_2.0.dna_rm.chromosome) and the associated gene annotation (Ustilago_maydis.Umaydis521_2.0.41.gff3; both downloaded from http://ftp.ensemblgenomes.org/pub/fungi/release-53/fasta/ustilago_maydis/dna/; 16). To incorporate potential 5´ UTR and 3´ UTR regions, which are not currently annotated in the Ustilago maydis genome, we manually extended all genes by 300 nucleotides (nt) on each side. This extension was determined based on the transcript read coverage profile (Fig. S13E-F; see Differential gene expression analysis). While the majority of UTRs were shorter than 150 nt, we observed a subset of transcripts with UTRs spanning 300 nt in length. To ensure comprehensive coverage of all potential editing sites, we decided to manually extend the genes by 300 nt on both sides of the open reading frame (ORF). The extended GTF file was converted to refFlat format using the UCSC gtfToGenePred tool.
+The A-to-G editing events on transcripts were identified using the wtRNA-RNA approach from the previously established hyperTRIBE pipeline (17; https://hypertribe.readthedocs.io/en/latest/run.html#b-find-rna-edit-sites-using-wtrna-rna-approaches). In brief, RNA-seq reads were trimmed based on sequencing quality (Phred score) using Trimmomatic (version 0.39; 18). Specifically, reads with a Phred score of less than 25 were trimmed and removed when the read length fell below 19 nt. The trimmed reads were then mapped to the U. maydis genome sequence and gene annotation using STAR (version 2.5.2b; 19), allowing reads that were mapped to exactly one location, had a minimum of 16 matched bases, and contained no more than 7% mismatches per mapped length. Uniquely mapped alignments in SAM format were loaded into a MySQL table with genomic coordinates. The nucleotides at each position in the test mRNA libraries (RBP-Ada-Gfp, control-Ada) were compared with those of the control mRNA library (wtRNA: RBP-Gfp) to identify A-to-G editing sites. Only editing events with a minimum of 20 read coverage and 5% editing were considered for further analysis. Editing sites that are assigned to more than one gene were removed from the analysis. From this, the list of reproducible editing sites that were consistently present in both replicates of either RBP-Ada-Gfp or control-Ada libraries was
+identified. The resulting reproducible editing events from RBP-Ada-Gfp and control-Ada were compared against each other to determine the editing sites that were unique to RBP-Ada-Gfp (Fig. S4A, S6A).
+Subcellular localization of proteins was defined using DeepLoc-2.0 (20; https://services.healthtech.dtu.dk/service.php?DeepLoc-2.0) and the cellular compartment ontology. Cellular compartment ontology annotations for U. maydis genes were downloaded from QuickGO (21, 22; https://www.ebi.ac.uk/QuickGO/annotations). A gene was assigned to more than one category if they were predicted to localize in more than one subcellular region.
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diff --git a/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/De novo motif discovery.md b/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/De novo motif discovery.md
new file mode 100644
index 0000000000000000000000000000000000000000..ee74df287bd41f6876f4d5b4f3d0864abe8902db
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+++ b/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/De novo motif discovery.md	
@@ -0,0 +1 @@
+For de novo motif discovery, we first extracted sequences by extending 250 nt on both sides of the editing sites using Biostrings (R package; version 2.56.0). We employed XSTREME from the MEME suite for motif discovery and enrichment analysis (23). Sequences of length 501 nt were randomly selected from the U. maydis genome and used as a background control (random genome background). XSTREME analysis was conducted using default parameters. The relative enrichment ratio (tested sequences vs. random genome background) of the identified motifs was compared against the absolute percentage of tested sequences with enriched motifs. Only motifs that exhibited both high enrichment and overrepresentation were considered potential RBP binding motifs. Motif logos were created using the ggseqlogo package (version 0.1) in R, utilizing the position-specific weight matrix (PWM) of the identified motifs.
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diff --git a/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/Motife analysis.md b/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/Motife analysis.md
new file mode 100644
index 0000000000000000000000000000000000000000..4d9798896c6bd51ce247139a844e5ad212a702b0
--- /dev/null
+++ b/assays/S3_A4_HyperTRIBE_motif_analysis/protocols/Motife analysis.md	
@@ -0,0 +1,4 @@
+The genomic position of the AUACCC, AGAUCU, GGGUAU, and ACACUC motifs on the U. maydis 521 genome sequences (PEDANT database name p3_t237631_Ust_maydi_v2GB) was identified using the Bioconductor package GenomicRanges in R (version 1.40.0; https://bioconductor.org/packages/release/bioc/html/GenomicRanges.html). Sequence information was extracted using Biostrings (R package; version 2.56.0; https://bioconductor.org/packages/release/bioc/html/Biostrings.html). To determine the genomic position of the de novo discovered motifs. The matchPWM function from the Biostrings R package was employed to perform the motif-matching process.
+To calculate the distance between hyperTRIBE editing sites and the nearest motif, we first obtained the genomic coordinates of editing sites. We then proceeded to determine the distance of each editing site to the nearest motif using the nearest-methods function in the GenomicRanges R package.
+To assess motif enrichment across transcript regions (Fig. 3F; Fig. S10A; Fig. S11A), we first determined the relative position of the motifs within the specific transcript regions, namely the 5´ UTR, ORF, and 3´ UTR. This was achieved by calculating the ratio between the distance from the motif start and the start codon to the length of the coding sequence (CDS). A ratio value ranging from 0 to 1 indicates a position within the ORF, a negative value suggests motif occurrence in the 5´ UTR, and a value greater than 1 suggests motif incidence in the 3´ UTR. For regions on the "-" strand, we multiplied the resulting position by -1, to appropriately represent the motif positions on the antisense strand, ensuring that the relative positions remained comparable between the two strands. Motif enrichment analysis was then carried out by computing the relative proportion of motifs within different transcript regions.
+For relative motif position calculation in SI Appendix, Fig. S8D, we first selected a window of 1000 nt with a start or stop codon at the center. We then obtained the genomic coordinates of the AUACCC and AGAUCU motifs within the selected window. The relative position of the motif on target transcripts was calculated by measuring the distance between the motif start position and the start or stop codon.
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diff --git a/assays/S3_A5_GO-term/protocols/GO term enrichment analysis.md b/assays/S3_A5_GO-term/protocols/GO term enrichment analysis.md
new file mode 100644
index 0000000000000000000000000000000000000000..b75c950770c402168ec33e6b2b8a65a3d0dd5b13
--- /dev/null
+++ b/assays/S3_A5_GO-term/protocols/GO term enrichment analysis.md	
@@ -0,0 +1 @@
+Functional enrichment analysis was performed in R using the R package gprofiler2 (e107_eg54_p17_bf42210; version 0.2.1; https://cran.r-project.org/web/packages/gprofiler2/vignettes/gprofiler2.html; 27, 28). For the g:Gost analysis, multiple testing correction was performed with the default g:SCS method and a p-value threshold of 0.05. Downloaded Generic EnrichmentMap (GEM) file from g:Profiler was loaded into the Cytoscape (version 3.8.0) plugin, EnrichmentMap (version 1.1.0) to visualize the GO term enrichment analysis (Jaccard coefficient cutoff of >0.25). The clusters were identified and annotated according to the GO-term annotation of the gene sets using the Cytoscape plugins AutoAnnotate (version 1.3.5) and the WorldCloud app (version 3.1.4) (29-31).
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diff --git a/assays/S4_A1_RNAseq_data/protocols/Differential gene expression analysis.md b/assays/S4_A1_RNAseq_data/protocols/Differential gene expression analysis.md
new file mode 100644
index 0000000000000000000000000000000000000000..c81c056431e6e2a32923c6e1c888fa2a53ff0e5b
--- /dev/null
+++ b/assays/S4_A1_RNAseq_data/protocols/Differential gene expression analysis.md	
@@ -0,0 +1,5 @@
+Total RNA from yeast and hyphal (9 h.p.i.) cells was extracted using RNeasy Plant Mini Kit (74904; Qiagen, Hilden, Germany) following the manufacturer’s protocol (see HyperTRIBE experiment and data processing). cDNA libraries were prepared using VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-01/02; Vazyme) and sequenced using the HiSeq 3000 platform (Illumina), to obtain 151-nt single-end reads. Approximately, ~10 million raw reads for hyphal and ~20 million raw reads for yeast samples were obtained.
+Basic quality control checks on all sequencing datasets were performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNAseq data analysis was carried out in Galaxy, an open-source platform (24). The raw sequencing reads were quality filtered based on their quality score and length with Trimmomatic (Version 0.36; 18). Specifically, the reads were trimmed 20 nt from the start, trimmed from the end when the Phred score dropped below 30, and discarded if the read length is less than 20 nt. STAR (version 2.7.2b) was used to align the trimmed reads to the U. maydis genome (see HyperTRIBE experiment and data processing; 19), allowing up to 4% mismatches in the mapped read length while limiting the mapping to one locus. Uniquely mapped reads on each gene were counted using htseq-count (version 0.9.1; 25). The resulting raw counts of mRNA libraries were used as input for the subsequent differential gene expression analysis. All differential gene expression analysis was performed using the R/Bioconductor package DESeq2 (26). Genes with an absolute fold change>1.5 (after fold change shrinkage) and an adjusted P<0.05 (Benjamini-Hochberg correction) were considered differentially expressed.
+To identify differentially expressed genes in hyphae following a morphological switch from yeast, quantitative changes in transcript expression levels were calculated by comparing the hyphal against the yeast mRNA libraries (“hyphae vs. yeast”) for wildtype as well as khd4 strains. As a result, transcripts that were more expressed in the hyphal form obtained a positive log2-transformed fold change (log2 fold change), while transcripts that were more expressed in the yeast form obtained a negative log2 fold change. To identify differentially expressed genes in the hyphal cells after khd4
+deletion, the mRNA libraries from the khd4 hyphal cells (9 h.p.i.) were compared against the mRNA libraries of wildtype hyphal cells (9 h.p.i.) (“khd4 hyphae vs. wildtype hyphae”).
+To identify potential UTR regions, the transcript read coverage profile was generated by analyzing the mapped BAM files (Fig. S13A). The coverage function from the IRanges package (R package version 2.32.0) was utilized to calculate the coverage of the mapped BAM file. Subsequently, each coverage value was normalized by dividing it by the total number of reads. To focus on regions surrounding the start or stop codons, we extracted the coverage values within a 501 nt window centered around these codons. These extracted values were then converted to z-scores by subtracting the mean of the respective row from each element in that row and dividing the result by the standard deviation of the row.
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diff --git a/assays/S6_A4_relative_fluorescence_level/protocols/Reporter fluorescence measurement.md b/assays/S6_A4_relative_fluorescence_level/protocols/Reporter fluorescence measurement.md
new file mode 100644
index 0000000000000000000000000000000000000000..273ae1b1ca8d4ff1df8eee518d388c763d47f08e
--- /dev/null
+++ b/assays/S6_A4_relative_fluorescence_level/protocols/Reporter fluorescence measurement.md	
@@ -0,0 +1 @@
+Hyphal growth was induced by shifting 20 ml of exponentially growing yeast cells in CM medium to NM medium, each supplemented with 1% glucose (promoter-off condition) or 1% arabinose (promoter-on condition). After 6 hours 1 ml of culture was harvested at 16000 g for 5 min at room temperature. Cell pellets were washed twice in double-distilled water. After harvesting, the supernatant was removed completely. The resulting cell pellets were resuspended using 1 ml of double-distilled water. 200 μl of samples were transferred into black 96-well plates (Greiner 96 Flat Bottom Black Polystyrene: Greiner, Frickenhausen, Germany). Optical density (OD600) and mKate2 fluorescence level (excitation wavelength: 588 nm, emission wavelength: 633 nm) were measured in an Infinite M200 plate reader (Tecan Group Ltd., Männedorf, Switzerland). The fluorescence levels (mKate2) were normalized to the optical density of the cell culture (OD600). The strain constitutively expressing mKate2 (kat_no motif) was used for calculating relative mKate2 protein abundance (Table S1). Statistical significance was calculated using multiple Student’s t-test in Prism version 5.04 (GraphPad, La Jolla, CA, USA).
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diff --git a/assays/S8_A1_microscopy_FM4-64/protocols/Staining technique FM4-64.md b/assays/S8_A1_microscopy_FM4-64/protocols/Staining technique FM4-64.md
new file mode 100644
index 0000000000000000000000000000000000000000..b91e974d5c8eb124391a029e8d33e16e7c960dad
--- /dev/null
+++ b/assays/S8_A1_microscopy_FM4-64/protocols/Staining technique FM4-64.md	
@@ -0,0 +1 @@
+Hyphal growth was induced in NM medium for 10 hours. 1 ml of culture was transferred into a 2 ml centrifuge tube and incubated for 10 min on ice. 4 μM (final concentration) of FM4-64 was added to the cells and incubated for an additional 10 min on ice, protected from light. Cells were washed by centrifugation at 2400 g for 3 min at 4 °C. Cell pellets were resuspended in 1 ml of ice-cold NM-glucose medium. Samples were shifted to a thermoblock set at 1100 rpm and 28 °C for 25 min prior to visualization under fluorescence microscopy.
diff --git a/assays/S8_A1_microscopy_FM4-64/protocols/imaging.md b/assays/S8_A1_microscopy_FM4-64/protocols/imaging.md
new file mode 100644
index 0000000000000000000000000000000000000000..0624294b255c51c1773f1ec1c506a96eff327446
--- /dev/null
+++ b/assays/S8_A1_microscopy_FM4-64/protocols/imaging.md
@@ -0,0 +1,3 @@
+To assess the FM4-64 (ThermoFisher) signal in hyphal cells, movies with an exposure time of 150 ms, and 100 frames were recorded. Kymographs from the movies were generated using the MetaMorph software package. Changes in direction were counted as an individual signal with the following parameters: a signal is processive if particles exhibit a directional travel distance of greater than 5 μm; diffusive if the signal traveled is less than 5 μm but greater than 0 μm. The diffusive particles display random
+14
+erratic movements without a clear trajectory. More than 25 hyphal cells were analyzed per strain (n=3 independent experiments).
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diff --git a/assays/S8_A2_microscopy_CMAG/protocols/Staining technique CMAG.md b/assays/S8_A2_microscopy_CMAG/protocols/Staining technique CMAG.md
new file mode 100644
index 0000000000000000000000000000000000000000..0260fa3d8cde310e1a4b843cf5e9cb55ca007abc
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+++ b/assays/S8_A2_microscopy_CMAG/protocols/Staining technique CMAG.md	
@@ -0,0 +1 @@
+Vacuolar staining was performed using CMAC (7-amino-4-chloromethylcoumarin; ThermoFisher, Darmstadt, Germany). 1 ml of cell suspension was stained with 10 μM CMAC and incubated for 30 min at 28 °C on a rotating wheel. Cells were washed by centrifugation at 2400 g for 3 min. Subsequently, the cell pellets were resuspended in phosphate-buffered saline prior to microscopy with the DAPI filter set.
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diff --git a/assays/S8_A2_microscopy_CMAG/protocols/imaging.md b/assays/S8_A2_microscopy_CMAG/protocols/imaging.md
new file mode 100644
index 0000000000000000000000000000000000000000..79bf01699f1ae2866a738319576bef0ba7bf2790
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+++ b/assays/S8_A2_microscopy_CMAG/protocols/imaging.md
@@ -0,0 +1 @@
+Vacuole distribution was scored by acquiring z-stacks (z-distance- 0.23 μm) and analyzing the maximum projection. The intensity profile of CMAC-stained hyphal cells was performed by line scan. Fluorescent intensity was plotted against their respective position after subtracting the background fluorescence of the micrograph. Vacuole distribution and aberrant cortical localization were scored by visual inspection (Fig. 7A-D). For the quantification of vacuole distribution, the line scans (see above; Fig. 7A, bottom panel) were used to distinguish normal and disrupted distribution. The cortical localization of vacuoles at the cell edge was quantified by visually counting the cells exhibiting this phenotype. For each strain, more than 25 cells were analyzed.
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