Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

RNA half-life is closely related to its cellular physiological function, so stability determinants may have regulatory functions. Micro(mi)RNAs have primarily been studied with respect to post-transcriptional mRNA regulation and target degradation. Here we study the impact of the tumour suppressive melanoma miRNA miR-211 on transcriptome stability and phenotype in the non-pigmented melanoma cell line, A375. Using 5ʹ-bromouridine IP chase (BRIC)-seq, transcriptome-wide RNA stability profiles revealed highly regulated genes and pathways important in this melanoma cell line. By combining BRIC-seq, RNA-seq and in silico predictions, we identified both existing and novel direct miR-211 targets. We validated DUSP3 as one such novel miR-211 target, which itself sustains colony formation and invasion in A375 cells via MAPK/PI3K signalling. miRNAs have the capacity to control RNA turnover as a gene expression mechanism, and RNA stability profiling is an excellent tool for interrogating functionally relevant gene regulatory pathways and miRNA targets when combined with other high-throughput and in silico approaches. Total RNA was isolated from A375 and A375/211 cell line for BRIC-seq and RNA-seq. For BRIC-seq, cells were labelled with BrU for 24 h and collected at 0, 1, 3 and 6 h post-labelling. For RNA-seq cells were collceted from A375 and A375/211 cell lines. Reads were aligned to hg38 genome using HISAT2 and quantified using StringTie. DESeq2 was used to perform differential gene expression analysis.