Figure 5. eIF3 Binding Sites within Cellular mRNAs Localize to Sites of m⁶A Residues within the 5′ UTR.

(A) Shown are read clusters from both eIF3 PAR-iCLIP (light blue) and single-nucleotide-resolution m⁶A mapping (Linder et al., 2015) (miCLIP; red) for four representative mRNAs (EIF4A3, H3F3C, SQLE, and IER5). eIF3a PAR-iCLIP read clusters exhibit highly specific overlap with m⁶A mapping clusters at internal positions within 5′ UTRs. This co-localization is specific to 5′ UTRs, as mRNAs that contain multiple m⁶A residues in the CDS or 3′ UTR fail to show eIF3a binding at these sites (exemplified by IER5). Red asterisks indicate the location of individual m⁶A sites identified at single-nucleotide resolution.

(B) eIF3 binds to the 5′ UTR of cellular mRNAs in an m⁶A-dependent manner. HEK293 cells were transfected with GFP- or Fto-overexpression plasmids, and eIF3 immunoprecipitation was performed to isolate eIF3-bound mRNAs. Bound mRNAs were quantified by RT-qPCR using 5′ UTR-specific primers. 5′ UTRs of mRNAs that contain high levels of m⁶A exhibited reduced binding to eIF3 after overexpression of Fto. 5′ UTRs that do not contain m⁶A exhibited no change in eIF3 binding following Fto overexpression (n = 3; mean ± SEM).

See also Figures S4 and S5.