Functional analysis of translation start sites based on ribosome profiling. (A) The CUG in ribosome footprint 3 was mutated to CCG and the plasmid was transfected into HEK cells for 24 h. Nano and firefly luciferase activities and nano and firefly RNA levels (by RT-qPCR) in the poly(GA) and poly(GP) frames were measured and their relative translational activities (activity to RNA) were plotted. (B) The CUGs in the three ribosome footprints were mutated to CCG and nano and firefly activities and their RNAs in the poly(GA) and poly(GP) frames were measured and plotted as above. (C) The AUG at the end of exon 1 was mutated to UAA and nano and firefly luciferase activities in the poly(GA) and poly(GP) frames were measured and plotted as above. (*) P < 0.05; (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001 (Student's t-test). (D) uORF formation and alignment of amino acid sequences of the uORF in human, orangutan, and macaque.
