5-methylcytosine (m5C) is an abundant RNA modification decorating diverse species of RNA. The re-discovery of m5C as an mRNA modification came to light by coupling bisulfite conversion of RNA with transcriptome-wide RNA sequencing (bsRNA-seq). Subsequent studies revealed prominence of deposition of m5C modification in regulating gene expression. Presently, bsRNA-seq experiments carried out in different cell types have generated several datasets for m5C-decorated mRNAs that are often different from each other. This disparity could be explained by differential methylation patterns in different cell types, the variation in the expression levels of the writer enzymes (NSUN family of proteins) and differences in data analysis pipelines. In an attempt to address this variability, we generated a comprehensive, transcriptome-wide human dataset of m5C sites by re-analysing existing bsRNA-seq datasets from 5 different human cell lines and 7 tissues, with a stringent pipeline for m5C site calling. This allowed us to compile a union list of 9,129 high-confidence m5C sites. This union list was then employed to investigate the relationship between m5C modification and RNA-binding proteins (RBPs) binding to mRNA. Therefore, we overlapped the exonic m5C sites with publicly available eCLIP data for >100 RBPs, identifying several proteins whose footprint significantly overlaps with m5C modification. We further generated a knockout HeLa cell line for NSUN2, the main m5C writer protein, to investigate the effect of mRNA methylation level and its influence on UPF1 RBP function. By performing UPF1 knockdown experiments, in WT vs NSUN2 knockout HeLa cells, we showed that the lack of NSUN2 enzyme reduces the effect of UPF1 in regulating the steady state of a subgroup of its methylated mRNA targets. CLIP experiments also showed a differential binding of UPF1 RBP depending on its targets methylation content. Overall, our data suggests an interplay between NSUN2 RNA methyltransferase activity and UPF1 function on mRNA.