RNA Modification Machinery Analysis

Scripts used to extract curated lists of RNA modification enzymes and assess their tissue-specificity across multiple species and tissues, as well as cancer and normal tissues, used in the paper:

Begik O, Lucas MC, Liu H, Ramirez JM, Mattick JS and Novoa EM. Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures. Genome Biology, May 2020. link: https://rdcu.be/b3Z5I. doi: https://doi.org/10.1186/s13059-020-02009-z

Data accompanying the paper can be found here HERE

PART1: Search and extract sets of RNA modification enzymes in selected species across the tree of life

FINDING RNA MODIFICATION ENZYMES in a specific SPECIES

Required: HMMER

find_homologs.sh <pfam_hmm> <fasta_reference_proteome> 

# Example: find_homologs.sh A_deamin.hmm.txt Saccharomyces_cerevisiae.fasta

• This script takes pfam profile as an input and fasta reference proteome
• Output will be proteins that have similiar functional domains, e.g. A_deamin.hmm.txtSaccharomyces_cerevisiae.fasta
• Manual curation is performed to select candidates (based on literature, etc)

ALIGNING THE ORTHOLOG PROTEINS AND BUILDING PHYLOGENETIC TREE FOR EACH GROUP OF ENZYMES

Extract fasta sequences for the proteins of interest in a list of orthologs proteins of a gene group

Required: perl

extract_fasta.sh <uniprot_ID_list>

# Example: extract_fasta.sh A_deamin

Align protein sequences with MAFFT align

Required: mafft

mafft.sh <FASTA>

# Example: mafft.sh uniprotIDlist.txt.named.fasta

Build build a phylogenetic tree with iqtree software using

Required : iqtree

iqtree.sh <MAFFT_OUTPUT>

# Example: iqtree.sh uniprotIDlist.txt.named.fasta.mafft

PART2: EXPRESSION ANALYSIS WITH HUMAN AND MOUSE DATASETS

EXTRACTING RNA MODIFICATION MACHINERY PROTEIN (RMP) EXPRESSION FROM RNA SEQ DATASETS

Initially obtained list of Main RNA Writer Proteins and we added non-catalytic subunits, readers, erasers and other tRNA writer proteins from V de Crécy-Lagard et al - 2019. Therefore, we have 146 RMMs at the end.

GTEx

Extract TPM values for the RNA modification enzymes from the GTEx TPM dataset

Rscript gtex_manipulation.R <GTEX.Expression.File> <ENSEMBL_GeneSymbol_Class.File>

# Example: Rscript gtex_manipulation.R GTEx_Analysis_2016-01-15_v7_RNASeQCv1.1.8_gene_median_tpm.gct human_id_symbol_class.tsv

ENCODE

Extract TPM values for the RNA modification enzymes from the ENCODE TPM dataset

Rscript encode_manipulation.R <ENCODE.Expression.File> <ENSEMBL_GeneSymbol_Class.File>

# Example: Rscript encode_manipulation.R mm65.long.gene.with.expr.cshl.tsv mouse_id_symbol_class.tsv

GTEx Tissue-Wide Expression Analysis

Scripts for tissue-specificity analysis and plots

Rscript gtex_tissuewide.R  <TPM table>

# Example: Rscript gtex_tissuewide.R RMLP.GTEX.TissueAveraged.TPM.tsv

ENCODE Tissue-Wide Expression Analysis

Scripts for tissue-specificity analysis and plots

Rscript encode_tissuewide.R  <TPM table>

# Example: Rscript encode_tissuewide.R RMLP.encode.TPM.brainav.tsv

Data similarity analysis between GTEx (Human) and ENCODE (Mouse)

Scripts for Pearson correlation analysis between two datasets

Rscript gtex_vs_encode_similarity.R  <GTEX data> <ENCODE data> 

# Example: Rscript gtex_vs_encode_similarity.R RMLP.GTEX.TissueAveraged.TPM.tsv RMLP.encode.TPM.brainav.tsv

PART3: EXPRESSION ANALYSIS WITH AMNIOTE AND PRIMATE SPECIES

Analysis for the Amniote Orthologs

Rscript kaessmann.amniote.R <input.expression.data> <ENSEMBL_GeneSymbol_Class.File>

# Example: Rscript kaessmann.amniote.R NormalizedRPKM_ConstitutiveExons_Amniote1to1Orthologues.txt human_id_symbol_class.tsv

Analysis for the Primate Orthologs

Rscript kaessmann.primate.R <input.expression.data> <ENSEMBL_GeneSymbol_Class.File>

# Example: Rscript kaessmann.primate.R NormalizedRPKM_ConstitutiveExons_Primate1to1Orthologues.txt human_id_symbol_class.tsv

PART4: EXPRESSION ANALYSIS IN SPERMATOGENESIS

Analysis of single-cell RNA sequencing data (Green et al., 2019)

Rscript spermatogenesis.R <spermatogenesis.expression.data> <ensembl_file>

# Example: Rscript spermatogenesis.R spermatogenesis_scRNA_averageexpression.tsv gene_hgnc_ensmus.tsv

PART5: TUMOR AND NORMAL TISSUE ANALYSIS

Extracting log(TPM+1) with reformatting

Rscript cancer_script1_datamanipulation.R <TCGA.GTEX.file> <ENSEMBL.file>

Example:  Rscript cancer_script1_datamanipulation.R RMLP.TcgaTargetGtex_rsem_gene_tpm_withheader.tsv human_id_symbol_class.tsv

Boxplots of individual patients (log)

Rscript cancer_script2_boxplot.R <TCGA.GTEX.file>

Example:  Rscript cancer_script2_boxplot.R TCGA_GTEX_FINAL.log2.without3cancer.tsv

Heatmap plot of average log(TPM) values

Rscript cancer_script3_mean_heatmap.R <TCGA.GTEX.file>

Example:  Rscript cancer_script3_mean_heatmap.R TCGA_GTEX_FINAL.log2.without3cancer.tsv

Calculation of log2FC values for RMPs in tumor/normal pairs and plots

Rscript cancer_script4_log2FC.R <MedianLog Tumor and Normal TPM file>  <Original TPM File>
 
Example: Rscript cancer_script4_log2FC.R medianlog_tumor_normal.tpm.tsv RMLP.TcgaTargetGtex_rsem_gene_tpm_withheader.tsv

Calculation of Dysregulation scores in tumor/normal pairs and plots

Rscript cancer_script5_Dysregulation.R <MedianLog Tumor and Normal TPM file for all genes> <MedianLog Tumor and Normal TPM file for RMPs>

Example:  Rscript cancer_script4_log2FC.R all_genes_logmedian_scatter_format.tsv medianlog_tumor_normal.tpm.tsv

Plotting the expression values with stage information

Rscript cancer_script6_Stage.R <Gene Expression File> <ENSEMBL_GeneSymbol_Class.File> <clinical information> <Phenotype data>

Example:  Rscript cancer_script6_stageexpression.R RMLP.TcgaTargetGtex_rsem_gene_tpm_withheader.tsv human_id_symbol_class.tsv clinical.tsv TcgaTargetGTEX_phenotype.txt

Plotting the survival curves

Rscript cancer_script7_SURVIVAL.R <Gene Expression File> <Survival data>

Example: Rscript cancer_script7_SURVIVAL.R TCGA_GTEX_FINAL.log2.without3cancer.tsv TCGA_survival_data