Fig. 2: Workflow of mRNA interactome capture using biotinylated oligo (dT) probes and identifying RBPs during bovine sperm capacitation.
From: Systematic mRNA interactome analysis reconceptualizes translational quiescence in bovine sperm
(1) Briefly, the physiological RBP-mRNA interaction was stabilized in vivo using UV crosslinking. (2) Sperm were lysed to release mRNA-RBP complex in the lysate (3) which was pulled down using streptavidin bead-conjugated biotinylated oligo (dT) probes hybridizing to poly A RNAs. (4) mRNA-RBP complex was eluted from beads by heat treatment. (5) The mRNA-RBP complex was treated with proteinase K to facilitate the recovery of mRNAs, which were then analyzed for nucleotide size distribution. The results were compared to total sperm RNA using an electrophoretic profile in a Bioanalyzer. Benzonase treatment digested mRNAs and recovered RBPs were visualized on a polyacrylamide gel using silver staining and identified using LC-MS/MS. Created in BioRender. Tiwari, S. (2025) https://BioRender.com/i9n9awi.
