Determine METTL16-mediated spatiotemoral installation of m6A via MeRIP-seq (m6A-seq) with nascent RNA and nuclear poly(A) RNA

METTL16 belong to methyltransferase like (METTL) family and possesses the ability to deposit N6-methyladenosine (m6A) in mRNA. We have conducted m6A-seq with poly(A) RNAs isolated from the whole HEK293T cells upon METTL16 knockdown to identify the transcripts with hypo m6A peaks after METTL16 knockdown, and also conducted RIP-seq with HEK293T cells to determine the METTL16-bound transcripts. However, we found that the transcripts with hypo m6A peaks upon METTL16 knockdown only account for a small proportion of METTL16-bound transcripts. We suppose one of the possibility reasons might be due to spatiotemporal installation of m6A. To support our hypothesis, we performed additional m6A seq with nascent RNA and nuclear poly(A) RNA isolated from HEK293T cells with METTL16 knockdown. The HEK293T cells were infected with shMETTL16-1, shMETTL16-2, and shNS lentivirus to induce knockdown of METTL16. The positive-infected cells were selected with 2ug/ml puromycin for 1-2 weeks. For purification of nascent RNA, all the cells were pulsed with 4-thiouridine (4sU) for 1 hours before collection. The nuclear pellets were collected with buffer RLN and then nuclear RNAs were extracted. The nascent RNAs were enriched and subjected to m6A IP. Poly(A) RNAs were enriched from nuclear RNAs and then subjected to m6A IP. Two different shRNAs against METTL16 were included to ensure the repeatibility and avoid potential off-target effects.