m⁶A Immunoprecipitation from Ribosome-Bound mRNA

Here we describe a protocol for correlating a transcript's translational state to its N6-methyladenosine (m⁶A) status using polysome profiling and m⁶A immunoprecipitation (IP). Polysome profiling is a technique used to separate out cellular components by density, allowing the visualization of transcripts based on the number of ribosomes bound. The technique uses high ribosome occupancy as a proxy for high translational activity. Transcripts bound by many ribosomes can be isolated from lowly translated transcripts and from those unbound by translational machinery. It has been demonstrated that the RNA modification m⁶A can alter the stability, localization, and splicing of a transcript. The role that m⁶A plays in translational selectivity is an established, but still highly debated area in the field. m⁶A IP is a technique developed to isolate m⁶A-containing RNA by using an antibody against the m⁶A modification itself. We have developed this protocol, which pairs polysome profiling with m⁶A IP in order to broadly characterize the relationship between the presence of m⁶A modifications and the extent of a transcript's translation.