Computational protocol: N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

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Protocol publication

[…] RNA-seq experiments were performed on two replicate RNA samples from HNRNPC, METTL3, METTL14 KD as well as control HEK293T cells (48 hours after transfection). Total RNA samples were extracted according to RNeasy plus kit (Catalog # 74104, Qiagen). Libraries were prepared according to the TruSeq Stranded mRNA LT Sample Prep Kit (Catalog # RS-122-9005DOC). KD and control samples were sequenced together in one flowcell on four lanes, respectively. All samples were sequenced by illumina Hiseq 2000 with pair end 100-bp read length. The reads from the four lanes of each sample were combined for all analysis. The RNA-seq data was mapped using the splice-aware alignment algorithm TopHat version 1.1.4 based on the following parameters: tophat –num-threads 8 –mate-inner-dist 200 –solexa quals –min-isoform-fraction 0 –coverage-search-segment-mismatches 1. Gene expression level changes were analyzed using cuffdiff. Differential splicing was determined using DEXSeq based on Cufflinks-predicted, nonoverlapping exons. To compare with a different mRNA binding protein, the RNA-seq data from HNRNPU KD HEK293T cells (GEO34995 dataset) was analyzed. [...] Gene Ontology (GO) enrichment analysis was applied on the co-regulated HCS-containing genes, against all HCS-containing genes as background, using GOrilla.Phylogenetic conservation analysis was performed by comparing PhyloP scores at the U-tracts motif and RRACH motif for hnRNP C m6A-switches to those of randomly selected sequences. The PhyloP scores were accessed from the precompiled phyloP scores (ftp://hgdownload.soe.ucsc.edu/goldenPath/hg19/phyloP46way/) under both primates and vertebrates categories. P-values were evaluated using the Mann-Whitney-Wilcoxon test, ***: p < 10 −16. For the U-tract motifs, we collected all U-tracts (5x U's) across all chromosomes and randomly selected 10,000 sites among the 38,561,577 sites of our census. The random selection was done separately for primates and for vertebrates. For the RRACH motif, we also collected all RRACH sites across all chromosomes and randomly selected 10,000 sites among the 78,815,225 sites of our census. Here too, the random selection was done separately for primates and vertebrates.Sequence logos were generated using the WebLogo package. R statistical package was used for all statistical analysis (unless stated otherwise). […]

Pipeline specifications

Software tools TopHat, Cufflinks, DEXSeq, GOrilla, PHAST, WebLogo
Applications Phylogenetics, RNA-seq analysis, Genome data visualization
Organisms Homo sapiens