FIG 11.

RIP-qRT-PCR measurement of RNA bound by YTHDC1, SRSF3, and SRSF10. (A) Schematic presentation of RIP–qRT-PCR procedure. (B) Locations of the primers used for qRT-PCR measurement of a protein-bound RNA fragment carrying m⁶A site A in ORF50 (RTA) pre-mRNA, as well as th eprimers used for detection of ORF50 (RTA) mRNA. (C) RIP products obtained by qRT-PCR analyzed in an agarose (2%) gel by electrophoresis. The ∼100-bp fragment (RTA mRNA) was detected only in the input (before RNase A digestion), suggesting that it was not protected by RNA binding proteins and, thus, was sensitive to RNase A digestion. In contrast, the ∼150-bp fragment (RTA pre-mRNA in the m⁶A site A region) was protected and could be pulled down by anti-YTHDC1 antibody but not control IgG.